Peptide derivatives

ABSTRACT

Pharmaceutical preparation containing an effective amount of acetylsalicylic acid and a compound of Formula I.
 
A 1 —A 2 —NH—(CH 2 ) n —B 
         wherein A 1 , A 2 , n and B are as defined in the specification, which is either the compound as such, or a stereoisomer thereof, and which may be in the form of a physiologically acceptable salt, in association with a pharmaceutical carrier. The combinations are suitable for use in inhibiting thrombin, or in the treatment or prophylaxis of thrombosis or hypercoagulability in patients in need thereof.

This application is a continuation of Ser. No. 09/398,826, filed Sep.17, 1999, now abandoned, which is a continuation of Ser. No. 08/382,036,filed Aug. 19, 1994, now abandoned, which is a 371 of PCT/SE94/00535filed Jun. 2, 1994, the entire content of which is hereby incorporatedby reference in this application.

This invention relates to new competitive inhibitors of trypsin-likeserine proteases, especially thrombin and kininogenases such askallikrein, their synthesis, pharmaceutical compositions containing thecompounds as active ingredients, and the use of the compounds asthrombin inhibitors and anticoagulants and as antiinflammatoryinhibitors, respectively.

The invention also relates to novel use of compounds as startingmaterials in synthesis of a serine protease inhibitor. Furthermore theinvention relates to a novel structural fragments in serine proteaseinhibitors.

BACKGROUND

Blood coagulation is the key process involved in both haemostasis (i.e.prevention of blood loss from a damaged vessel) and thrombosis (i.e. thepathological occlusion of a blood vessel by a blood clot). Coagulationis the result of a complex series of enzymatic reactions, where one ofthe final steps is conversion of the proenzyme prothrombin to the activeenzyme thrombin.

Thrombin plays a central role in coagulation. It activates platelets, itconverts fibrinogen into fibrin monomers, which polymerise spontaneouslyinto filaments, and it activates factor XIII, which in turn crosslinksthe polymer to insoluble fibrin. Thrombin further activates factor V andfactor VIII in a positive feedback reaction. Inhibitors of thrombin aretherefore expected to be effective anticoagulants by inhibition ofplatelets, fibrin formation and fibrin stabilization. By inhibiting thepositive feedback mechanism they are expected to excert inhibition earlyin the chain of events leading to coagulation and thrombosis.

Kininogenases are serine proteases that act on kininogens to producekinins (bradykinin, kallidin, and Met-Lys-bradykinin). Plasmakallikrein, tissue kallikrein, and mast cell tryptase representimportant kininogenases.

Kinins (bradykinin, kallidin) are generally involved in inflammation.For example, the active inflammation process is associated withincreased permeability of the blood vessels resulting in extravasationof plasma into the tissue. The ensuing plasma exudate contains all theprotein systems of circulating blood. The plasma-derived kininogensinevitably will be interacting with different kallikreins, formingkinins continually as long as the active plasma exudation process isongoing. Plasma exudation occurs independent of the mechanisms that areinvolved in the inflammation, whether it is allergy, infection or otherfactors (Persson et al., Editorial, Thorax, 1992, 47:993-1000). Plasmaexudation is thus a feature of many diseases including asthma, rhinitis,common cold, and inflammatory bowel diseases. Particulary in allergymast cell tryptase will be released (Salomonsson et al., Am. Rev.Respir. Dis., 1992, 146:1535-1542) to contribute to kinin formation andother pathogenic events in asthma, rhinitis, and intestinal diseases.

The kinins are biologically highly active substances with smooth muscleeffects, sectretory effects, neurogenic effects, and actions that mayperpetuate inflammatory processes including activation of phospholipaseA₂ and increasing vascular permeability. The latter action potentiallyinduces a vicious circle with kinins providing for the generation ofmore kinins etc.

Tissue kallikrein cleaves primarily low molecular weight kininogen toproduce kallidin and plasma kallikrein preferably releases bradykininfrom high molecular weight kininogen.

PRIOR ART

Inhibitors of thrombin based on the amino acid sequence around thecleavage site for the fibrinogen Aα chain were first reported byBlombäck et al. in J. Clin. Lab. Invest. 24, suppl 107, 59, (1969), whosuggested the sequence Phe-Val-Arg (P9-P2-P1, herein referred to as theP3-P2-P1 sequence) to be the best inhibitor.

In U.S. Pat. No. 4,346,078 has S. Bajusz et al. described the thrombininhibitor H-DPhe-Pro-Agm, a dipeptidyl derivative with an aminoalkylguanidine in the P1-position.

Inhibitors of thrombin based on peptide derivatives with a cyclicaminoalkyl guanidine, e.g. 3-aminomethyl-1-amidinopiperidine, in theP1-position have been disclosed in EP-A2-0,468,231.

In EP-A2-0,185,390 has S. Bajusz et. al. disclosed that replacing theagmatine with an arginine aldehyde gave a thrombin inhibitor which hadmuch higher potency.

Inhibitors of kallikrein based on the amino acid sequence around thecleavage site Arg-Ser have been reported earlier.

The arginine chloromethyl ketones H-DPro-Phe-Arg-CH₂Cl and H-DPhe-Phe-Arg-CH₂Cl were reported as plasma kallikrein inhibitors byKettner and Shaw in Biochemistry 1978, 17:4778-4784 and Meth. Enzym.1981, 80:826-842.

Likewise, esters and amides containing the H-DPro-Phe-Arg sequence werereported by Fareed et al. in Ann. N.Y. Acad. Sci. 1981, 370:765-784 tobe plasma kallikrein inhibitors.

Inhibitors of serine proteases that are based on electrophilic ketonesinstead of aldehydes in the P1-position are described in the followingpatent documents:

EP-A2-0,195,212 describing peptidyl α-keto esters and amides,EP-A1-0,362,002 describing fluoroalkylamide ketones and EP-A2-0,364,344describing α,β,δ-triketo compounds possessing different peptidaseinhibiting properties.

Inhibitors of trypsin-like serine proteases, such as thrombin andkallikrein, based on C-terminal boronic acid derivatives of arginine andisothiouronium analogues thereof have been revealed in EP-A2-0,293,881.

WO 92/04371 describing kininogenase inhibitors, e.g. kallikreininhibitors based on derivatives of arginine.

EP-A1-0,530,167 describing α-alkoxy ketone derivatives of arginine asthrombin inhibitors.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide novel and potenttrypsine-like serine protease inhibitors, especially anticoagulantia andantiinflammatory compounds with competitive inhibitory activity towardstheir enzyme i.e. causing reversible inhibition. More specificallyanticoagulants for prophylaxis and treatment of thromboembolic diseasessuch as venous thrombosis, pulmonary embolism, arterial thrombosis, inparticular myocardial infarction and cerebral thrombosis, generalhypercoagulable states and local hypercoagulable states, e.g. followingangioplasty and coronary bypass operations, and other situations wherethrombin is believed to play a role, e.g. Alzheimers disease, as well asinhibition of kininogenases for treatment of inflammatory disorders e.g.asthma, rhinitis, urticaria, inflammatory bowel disease, and arthritis.A further object is to obtain thrombin inhibitors which are orallybioavailable and selective in inhibiting thrombin over other serineproteases. A further object of the invention is to obtain kininogenaseinhibitors which can be given orally, rectally, topically e.g. dermally,or via the inhalation route.

Compounds

According to the invention it has been found that compounds of thegeneral Formula I, either as such or in the form of physiologicallyacceptable salts, and including stereoisomers, are potent inhibitors ofserine proteases, especially thrombin and kininogenases such askallikrein:

wherein:

-   A¹ represents a structural fragment of Formula IIa, IIb, IIc, IId or    IIe;    wherein:-   k is an integer 0, 1, 2, 3 or 4;-   m is an integer 0, 2, 3 or 4;-   q is an integer 0, 1, 2 or 3;-   R¹ represents H, an alkyl group having 1 to 4 carbon atoms, or    R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is    possibly substituted in the position which is alpha to the carbonyl    group, and the alpha substituent is a group R¹⁷—(CH₂)_(p)—, wherein    p is 0, 1 or 2 and R¹⁷ is methyl, phenyl, OH, COOR¹², CONHR¹², where    R¹² is H or an alkyl group having 1 to 4 carbon atoms, and R¹¹ is H    or an alkyl group having 1 to 6 carbon atoms, or-   R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or-   R¹ represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4    carbon atoms and is possibly substituted alpha to the carbonyl with    an alkyl group having 1 to 4 carbon atoms and where R¹³ is H or an    alkyl group having 1 to 4 carbon atoms or —CH₂COOR¹², where R¹² is    as defined above, or-   R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to    4 carbon atoms and is possibly substituted alpha to the carbonyl    with an alkyl group having 1 to 4 carbon atoms and where R¹² is as    defined above, or-   R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,    Ph(2-COOR¹²)—O₂—, where R¹² is as defined above and R¹⁴ is an alkyl    group having 1-4 carbon atoms, or-   R¹ represents —CO—R¹⁵, wherein R's is an alkyl group having 1-4    carbon atoms, or-   R¹ represents —CO—OR¹⁵, where R¹⁵ is as defined above, or-   R¹ represent —CO—(CH₂)_(p)—COOR¹², where R¹² is as defined above and    p is an interger 0, 1 or 2, or-   R¹ represents —CH₂PO(OR¹⁶)₂, —CH₂SO₃H or —CH₂-(5-(1H)-tetrazolyl),    where R¹⁶ is, individually at each occurrence, H, methyl or ethyl;-   R² represents H or an alkyl group having 1 to 4 carbon atoms or    R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and,    where R²¹ is H or an alkyl group having 1 to 4 carbon atoms;-   R³ represents an alkyl group having 1-4 carbon atoms, and the alkyl    group may or may not carry one or more flourine atoms, or-   R³ represents a cyclopentyl, cyclohexyl- or a phenyl group which may    or may not be substituted with an alkyl group having 1 to 4 carbon    atoms, or-   R³ represents a phenyl group substituted with a OR³¹ group, where    R³¹ is H or an alkyl group having 1 to 4 carbon atoms and k is 0, 1,    or-   R³ represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or-   R³ represent a cis- or trans-decalin group and k is 0, 1, or-   R³ represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl which may or may    not be substituted with a OR³¹ group, where R³¹ is as defined above    and k is 0, 1, or-   R³ represents Si(Me)₃ or CH(R³²)₂, wherein R³² is a cyclohexyl- or a    phenyl group;-   R⁴ represents H, an alkyl group having 1 to 4 carbon atoms, a    cyclohexyl- or a phenyl group;-   A² represents a structural fragment of Formula IIIa, IIIb or IIIc    wherein:-   p is an interger 0, 1 or 2;-   m is an integer 1, 2, 3 or 4;-   Y represents a methylene group, or-   Y represents an ethylene group and the resulting 5-membered ring may    or may not carry one or two fluorine atoms, a hydroxy group or an    oxo group in position 4, or may or may not be unsaturated, or-   Y represents —CH₂—O—, —CH₂—S—, —CH₂—SO—, with the heteroatom    functionality in position 4, or-   Y represents a n-propylene group and the resulting 6-membered ring    may or may not carry in position 5 one fluorine atom, a hydroxy    group or an oxo group, carry two fluorine atoms in one of positions    4 or 5 or be unsaturated in position 4 and 5, or carry in position 4    an alkyl group with 1 to 4 carbon atoms, or-   Y represents —CH₂—O—CH₂—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—, or-   Y represent —CH₂—CH₂—CH₂—CH₂—;-   R³ is as defined above;-   R⁵ represents H or an alkyl group having 1 to 4 carbon atoms, or-   R⁵ represents —(CH₂)_(n)—COOR⁵¹, where p is 0, 1 or 2 and R⁵¹ is H    or an alkyl group having 1 to 4 carbon atoms;-   n is an integer 0, 1, 2, 3 or 4;-   B represents a structural fragment of Formula IVa, IVb, IVc or IVd    wherein:-   r is an interger 0 or 1;-   X¹ represent CH₂, NH or is absent;-   X² represents CH₂, NH or C═NH;-   X³ represents NH, C═NH, N—C(NH)—NH₂, CH—C(NH)—NH₂, CH—NH—C(NH)—NH₂    or CH—CH₂—C(NH)—NH₂;-   X⁴ represents CH₂ or NH;

Preferred combinations of X¹, X², X³, 4 and r are

-   X¹, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 0, 1, or,-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0, 1, or-   X¹ and X³ are NH, X² is C—NH, X⁴ is CH₂ and r is 0, 1, or-   X¹ and X⁴ are CH₂, X² is C═NH, X³ is NH and r is 0, 1, or-   X¹ is CH₂, X² and X⁴ are NH, X³ is C═NH and r is 1, or-   X¹, X² and X⁴ are CH₂, X³ is CH—NH—C(NH)—NH₂ and r is 0, 1, or-   X¹ is absent, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 0, or-   X¹ is absent, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0;    Particularly preferred combinations of X¹, X², X³, X⁴ and r are-   X¹, X² and X⁴ are CH₂. X³ is CH—C(NH)—NH₂ and r is 1;-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0 or-   X¹ is absent, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0;-   X¹ and X³ are NH, X² is C═NH, X⁴ is CH₂ and r is 1;-   X⁵ represents C(NH)—NH₂ or NH—C(NH)—NH₂;-   R⁶ is H or an alkyl group having 1-4 carbon atoms;-   X⁶ represents CH or N;

Compounds of Formula I having S-configuration on the A² amino acid arepreferred ones, of those compounds also having R-configuration on the A¹amino acid are particularly preferred ones.

In the present context the term “an alkyl group having 1 to 4 carbonatoms” may be straight or branched unless specified otherwise. An alkylgroup having 1 to 4 carbon atoms may be methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl and t-butyl.

In the present context the term “an alkyl group having 1 to 6 carbonatoms” may be straigh or branched unless specified otherwise. An alkylgroup having 1 to 6 carbon atoms may be methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl,t-pentyl, neo-pentyl, n-hexyl or i-hexyl. When unsaturation is referredto, a carbon-carbon double bond is intended.

The wavy lines on the carbon atom in the carbonyl group in formulas IIa,IIb, IIc, IId, IIe, IIIa, IIIb, IIIc, on the nitrogen atom in formulasIIIa, IIIb, IIIc and on the carbon atom in the ring system in formulasIVa, IVb, IVc, IVd signify the bond position of the fragment.

Abbreviations are listed at the end of this specification.

According to the invention it has been found that compounds of thegeneral Formula Ia, either as such or in the form of physiologicallyacceptable salts, and including stereoisomers, are potent inhibitors ofthrombin:

wherein:

-   A¹ represents a structural fragment of Formula IIa, IIb, IIc or IId,    preferably IIa or IIb;    wherein:-   k is an integer 0, 1, 2, 3 or 4, preferably 0, 1;-   q is an integer 0, 1, 2 or 3, preferably 1;-   R¹ represents H, an alkyl group having 1 to 4 carbon atoms,    R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is    possibly substituted in the position which is alpha to the carbonyl    group, and the alpha substituent is a group R¹⁷—(CH₂)_(n)—, wherein    p is 0, 1 or 2 and R¹⁷ is methyl, phenyl, OH, COOR¹², CONHR¹², where    R¹² is H or an alkyl group having 1 to 4 carbon atoms, and R¹¹ is H    or an alkyl group having 1 to 6 carbon atoms, or-   R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or-   R¹ represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4    carbon atoms and is possibly substituted alpha to the carbonyl with    an alkyl group having 1 to 4 carbon atoms and where R¹³ is H or an    alkyl group having 1 to 4 carbon atoms or —CH₂COOR¹² where R¹² is as    defined above, or-   R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to    4 carbon atoms and is possibly substituted alpha to the carbonyl    with an alkyl group having 1 to 4 carbon atoms and where R¹² is as    defined above, or-   R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,    Ph(2-COOR¹²)—SO₂— where R¹² is as defined above and R¹⁴ is an    alkylgroup having 1-4 carbon atoms, or-   R¹ represents —CO—R¹⁵, wherein R¹⁵ is an alkyl group having 1-4    carbon atoms, or-   R¹ represents —CO—OR¹⁵, where R¹⁵ is as defined above, or-   R¹ represent —CO—(CH₂)_(p)—COOR¹², where R¹² is as defined above and    p is an interger 0, 1 or 2, or-   R¹ represents —CH₂PO(OR¹⁶)₂, —CH₂SO₃H or —CH₂-(5-(1H)-tetrazolyl),    where R¹⁶ is, individually at each occurrence, H, methyl or ethyl;    Preferably R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1    to 4 carbon atoms and R¹¹ is H.-   R² represents H or an alkyl group having 1 to 4 carbon atoms, or    R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R²¹    is H or an alkyl group having 1 to 4 carbon atoms;-   R³ represents an alkyl group having 1-4 carbon atoms, and the alkyl    group may or may not carry one or more fluorine atoms, or-   R³ represents a cyclopentyl, cyclohexyl- or a phenyl group which may    or may not be substituted with an alkyl group having 1 to 4 carbon    atoms, or-   R³ represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or-   R³ represent a cis- or trans-decalin group and k is 0, 1, or-   R³ represents Si(Me)₃ or CH(R³²)₂₁ wherein R³² is a cyclohexyl- or    phenyl group;-   R⁴ represents an alkyl group having 1 to 4 carbon atoms, a    cyclohexyl or a phenyl group, preferably a cyclohexyl or a phenyl    group;-   A² represents a structural fragment of Formula IIIa, IIIb or IIIc,    preferably IIIa;    wherein:-   p is an interger 0, 1 or 2;-   m is an integer 1, 2, 3 or 4, preferably 2, 3;-   Y represents a methylene group, or-   Y represents an ethylene group and the resulting 5-membered ring may    or may not carry one or two fluorine atoms, a hydroxy group or an    oxo group in position 4, or may or may not be unsaturated, or-   Y represents —CH₂—O—, —CH₂—S—, —CH₂—SO—, with the heteroatom    functionality in position 4, or-   Y represents a n-propylene group and the resulting 6-membered ring    may or may not carry in position 5 one fluorine atom, a hydroxy    group or an oxo group, carry two fluorine atoms in one of positions    4 or 5 or be unsaturated in position 4 and 5, or carry in position 4    an alkyl group with 1 to 4 carbon atoms, or-   Y represents —CH₂—O—CH₂—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—, or-   Y represent —CH₂—CH₂—CH₂—CH₂—;-   R³ represents an alkyl group having 1-4 carbon atoms, or-   R³ represents a Si(Me)₃ group;-   R⁵ represents H or an alkyl group having 1 to 4 carbon atoms,    preferably H or a methylgroup, or-   R⁵ represents —(CH₂)_(p)—COOR⁵¹, where p is 0, 1 or 2 and R⁵¹ is H    or an alkyl group having 1 to 4 carbon atoms, preferably p is 0 and    R⁵¹ is H;-   n is an integer 0, 1, 2, 3 or 4, preferably 1, 2, 3;-   B represents a structural fragment of Formula IVa, IVb, IVc or IVd,    preferably IVa or IVb    wherein:-   X¹, X², X³, X⁴, X⁵ and X⁶ are as defined above;-   r is an integer 0 or 1;-   R⁶ is H or an alkyl group having 1-4 carbon atoms, preferably H;    preferred combinations of X¹, X², X³, X⁴ and r are-   X¹, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 0 or 1, or-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0 or 1, or-   X¹ and X³ are NH, X² is C═NH, X⁴ is CH₂ and r is 0 or 1, or-   X¹ and X⁴ are CH₂′, X² is C═NH, X³ is NH and r is 0 or 1, or-   X¹ is CH₂′ X² and X⁴ are NH, X³ is C═NH and r is 1, or-   X¹, X² and X⁴ are CH₂, X³ is CH—NH—C(NH)—NH₂ and r=0 or 1, or-   or X¹ is absent, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 0,-   or X¹ is absent, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0;    Particularly preferred combinations of X¹, X², X³, X⁴ and r are-   X¹ is absent, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0, or-   X¹, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r=1, or-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r=0 or 1, or-   X¹ and X³ are NH, X² is C═NH, X⁴ is CH₂ r is 1;-   X⁵ represents C(NH)—NH₂ or NH—C(NH)—NH₂, preferably C(NH)—NH₂;-   X⁶ represents CH or N;    According to a preferred embodiment the invention relates to    compounds of Formula Ia,    wherein:-   A¹ represents a structural fragment of Formula IIa,    wherein:-   k is 0 or 1;-   R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon    atoms, particularly methylene, ethylene and R¹¹ is H;-   R² represents H;-   R³ represents a cyclohexyl group;-   A² represents a structural fragment of Formula IIIa, wherein:-   Y represents a methylene group, an ethylene group, or a n-propylene    group and the resulting 6-membered ring may or may not carry in    position 4 an alkyl group with 1 to 4 carbon atoms, preferably Y    represents methylene, ethylene;-   R⁵ represents H;-   B represents a structural fragment of formula IVa wherein:-   X¹ is absent, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂, r is 0 and n is    1 or 2;-   X¹, and X³ are NH, X² is C═NH, X⁴ is CH₂, r is 1 and n is 2, or-   X¹, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂, r is 1 and n is 1, or-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂, r is 0 or 1 and n is 1 or    2, or

More particularly preferred are compounds wherein B represents astructural fragment for formula IVb wherein:

-   X⁵ represents C(NH)—NH₂, R⁶ is H, and n=1

Preferred compounds of the invention are:

-   HOOC—CH₂—(R)Cgl-Aze-Pab-   HOOC—CH₂—CH₂—(R)Cgl-Aze-Pab-   HOOC—CH₂—(R)Cgl-Pro-Pab-   HOOC—CH₂—CH₂—(R)Cgl-Pro-Pab-   (HOOC—CH₂)₂—(R)Cgl-Pro-Pab-   H—(R)Cgl-Pic-Pab-   HOOC—CH₂—(R,S)CH(COOH)—(R)Cgl-Pic-Pab-   H—(R)Cha-Aze-Pab-   HOOC—CH₂—(R)Cha-Aze-Pab-   HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Aze-Pab-   HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Aze-Pab/a-   HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Aze-Pab/b-   HOOC—CH₂—CH₂—(R)Cha-Aze-Pab-   HOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab-   H—(R)Cha-Pro-Pab-   HOOC—CH₂—(R)Cha-Pro-Pab-   HOOC—CH₂—(Me)(R)Cha-Pro-Pab-   HOOC—CH₂—CH₂—(R)Cha-Pro-Pab-   HOOC—CH₂—CH₂-(Me)(R)Cha-Pro-Pab-   HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pro-Pab/a-   HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pro-Pab/b-   HOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab-   EtOOC—CH₂—CH₂—CH₂—(R)Cha-Pro-Pab-   Ph (4-COOH)—SO₂—(R)Cha-Pro-Pab-   H—(R)Cha-Pic-Pab-   HOOC—CH₂—(R)Cha-Pic-Pab-   HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pic-Pab/a-   HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pic-Pab/b-   HOOC—CH₂—CH₂—(R) cha-Pic-Pab-   HOOC—CO—(R)Cha-Pic-Pab-   HOOC—CH₂—CO—(R) cha-Pic-Pab-   Me—OOC—CH₂—CO—(R)Cha-Pic-Pab-   H₂N—CO—CH₂—(R)Cha-Pic-Pab-   Boc-(R)Cha-Pic-Pab-   Ac—(R)Cha-Pic-Pab-   Me—SO₂—(R)Cha-Pic-Pab-   H—(R)Cha-(R,S)betaPic-Pab-   HOOC—CH₂—CH₂—(R)Cha-(R,S) betapic-Pab-   HOOC—CH₂—(R)Cha-Val-Pab-   HOOC—CH₂—CH₂—(R)Cha-Val-Pab-   H—(R)Hoc-Aze-Pab-   HOOC—CH₂—CH₂—(R)Hoc-Aze-Pab-   HOOC—CH₂—(R,S)CH(COOH)—(R)Hoc-Pro-Pab-   HOOC—CH₂—(R)Hoc-Pic-Pab-   (HOOC—CH₂)₂—(R)Hoc-Pic-Pab-   HOOC—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab-   HOOC—CH₂—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab-   HOOC—CH₂—CH₂—(R)Tic-Pro-Pab-   HOOC—CH₂—CH₂—(R)Cgl-Aze-Pig-   HOOC—CH₂—(R)Cgl-Pro-Pig-   H—(R)Cha-Aze-Pig-   HOOC—CH₂—(R)Cgl-Aze-Pac-   H—(R)Cha-Pro-Pac-   H—(R)Cgl-Ile-Pab-   H—(R)Cgl-Aze-Pab-   HOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab-   MeOOC—CH₂—(R)Cgl-Aze-Pab-   EtOOC—CH₂—(R)Cgl-Aze-Pab-   ^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab-   ^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab-   H—(R)Cgl-Pro-Pac-   HOOC—CH₂—(R)Cha-Pro-Pac-   HOOC—CH₂—CH₂—(R)Cgl-Pro-Pac-   HOOC—CH₂—CH₂—(R)Cha-Aze-Pac-   HOOC—CH₂—(R)cha-Aze-Pig-   HOOC—CH₂—(R)Cha-Pro-Pig-   HOOC—CH₂—CH₂—(R)Cha-Pro-Pig-   (HOOC—CH₂)₂—(R)Cgl-Pro-Pig-   HOOC—CH₂—CH₂(HOOC—CH₂)—(R)Cha-Pro-Pig-   HOOC—CH₂—(R)Cgl-Aze-(R,S)Itp-   HOOC—CH₂—(R)Cha-Aze-(R,S)Itp-   H—(R)Cha-Pic-(R,S)Itp-   HOOC—CH₂—(R)Cha-Pic-(R,S)Itp-   H—(R)Cgl-Pro-(R,S)Hig-   HOOC—CH₂—(R)Cgl-Pro-(R,S)Hig-   H—(R)Cha-Pro-(R,S)Hig-   H—(R)Cgl-Aze-Rig-   HOOC—CH₁₂—(R)Cgl-Aze-Rig-   HOOC—CH₂—(R)Cha-Pro-Rig-   HOOC—CH₂—CH₂—(R)Cha-Aze-Rig-   HOOC—CH₂—(R)Cha-Pro-(S)Itp-   H—(R)Cha-Pro-(R,S)Nig-   H—(R)Cha-Pro-Mig-   H—(R)Cha-Pro-Dig-   H—(R)Cha-Aze-Dig

At present the particularly preferred compounds of formula Ia is

-   HOOC—CH₂—(R)Cgl-Aze-Pab-   HOOC—CH₂—CH₂—(R)Cha-Aze-Pab-   HOOC—CH₂—(R)Cha-Pro-Pab-   HOOC—CH₂—CH₂—(R)Cha-Pro-Pab-   HOOC—CH₂—(R)Cha-Pic-Pab-   HOOC—CH₂—(R)Cgl-Pro-Pig-   EtOOC—CH₂—(R)Cgl-Aze-Pab-   HOOC—CH₂—(R)Cha-Pro-Pac-   HOOC—CH₂—(R)Cha-Pro-Pig

In the above tables of compounds, the letters /a and /b refer to asubstantially pure stereoisomer at the carbon atom noted “R or S”. Thestereoisomer can be identified for each compound with reference to theexperimental part herein. “R,S” refers to a mixture of stereoisomers.

According to the invention it has been found that compounds of thegeneral Formula Ib, either as such or in the form of physiologicallyacceptable salts, and including stereoisomers, are potent inhibitors ofkininogenases:A¹—A²—NH—(CH₂)_(n)—B  Ibwherein:

-   A¹ represents a structural fragment of formula IIa, IIb or IIe,    preferably IIa or IIb;    wherein:-   k is an integer 0, 1, 2, 3 or 4, preferably 0, 1;-   q is an integer 0, 1, 2, or 3, preferably 1;-   R¹ represents H, an alkyl group having 1 to 4 carbon atoms, or    R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and is    possibly substituted in the position which is alpha to the carbonyl    group, and the alpha substituent is a group R¹⁷—(CH₂)_(p)—, wherein    p is 0, 1 or 2 and R¹⁷ is methyl, phenyl, OH, COOR¹², CONHR¹², where    R¹² is H or an alkyl group having 1 to 4 carbon atoms, and R¹¹ is H    or an alkyl group having 1 to 6 carbon atoms, or-   R¹ represents Ph(4-COOR²)—CH₂—, where R¹² is H or an alkyl group    having 1 to 4 carbon atoms, or-   R¹ represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4    carbon atoms and is possibly substituted alpha to the carbonyl with    an alkyl group having 1 to 4 carbon atoms and where R¹³ is H or an    alkyl group having 1 to 4 carbon atoms or —CH₂COOR¹² where R¹² is as    defined above, or-   R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to    4 carbon atoms and is possibly substituted alpha to the carbonyl    with an alkyl group having 1 to 4 carbon atoms and where R¹² is as    defined above, or-   R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂,    Ph(2-COOR¹²)—SO₂—, where R¹² is as defined above and R¹⁴ is an    alkylgroup having 1-4 carbon atoms, or-   R¹ represents —CO—R¹⁵, wherein R¹⁵ is an alkyl group having 1-4    carbon atoms, or-   R¹ represents —CO—OR¹⁵, where R's is as defined above, or-   R¹ represent —CO—(CH₂)_(n)—COOR¹², where R¹² is as defined above and    p is 0, 1 or 2, or-   R¹ represents —CH₂PO(OR¹⁶)₂, —CH₂SO₃H or —CH₂-(5-(1H)-tetrazolyl),    where R¹⁶ is, individually at each occurrence, H, methyl or ethyl;-   R² represents H or an alkyl group having 1 to 4 carbon atoms or    R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R²¹    is H or an alkyl group having 1 to 4 carbon atoms;-   R³ represents an alkyl group having 1-4 carbon atoms, and the alkyl    group may or may not carry one or more fluorine atoms, or-   R³ represents a cyclopentyl, cyclohexyl- or a phenyl group which may    or may not be substituted with an alkyl group having 1 to 4 carbon    atoms, or-   R³ represents a phenyl group substituted with a OR³¹ group, where    R³¹ is H or an alkyl group having 1 to 4 carbon atoms and k is 0, 1,    or-   R³ represents a 1-naphthyl or 1-naphthyl group and k is 0, 1, or-   R³ represent a cis- or trans-decalin group and k is 0,1, or-   R³ represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl which may or may    not be substituted with a OR³¹ group, where R³¹ is as defined above    and k is 0, 1, or-   R³ represents Si(Me)₃ or CH(R³²)₂, wherein R³² is a cyclohexyl- or    phenyl group;-   R⁴ represents H, an alkyl group having 1 to carbon atoms, a    cyclohexyl or a phenyl group, preferably H;-   A² represents a structural fragment of formula IIIb or IIIc,    preferably IIIb    wherein:-   p is an integer 0, 1 or 2;-   m is an integer 1, 2, 3, or 4, preferably 2, 3;-   R³ is as defined above;-   n is an integer 0, 1, 2, 3 or 4, preferably 1,2,3;-   B represents a structural fragment of Formula IVa, IVb, IVc or IVd,    preferably IVa or IVb;    wherein:-   X¹, X², X³, X⁴ are as defined above;-   R⁶ is H or an alkyl group having 1-4 carbon atoms, preferably H or a    methyl group;-   r is an integer 0 or 1;-   preferred combinations of X¹, X², X³ and X⁴ are-   X¹, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 0 or 1, or-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0 or 1, or-   X¹ and X³ are NH, X² is C═NH, X⁴ is CH₂ and r is 0 or 1, or-   X¹ and X⁴ are CH₂, X² is C═NH, X³ is NH and r is 0 or 1, or-   X¹ is CH₂, X² and X⁴ are NH, X³ is C═NH and r is 1, or-   X¹, X² and X⁴ are CH₂, X³ is CH—NH—C(NH)—NH₂ and r is 0 or 1,-   or X¹ is absent, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 0,    or-   X¹ is absent, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 0;    particularly preferred combinations of X¹, X², X³ and X⁴ are-   X¹, X² and X⁴ are CH₂, X³ is CH—C(NH)—NH₂ and r is 1 or,-   X¹, X² and X⁴ are CH₂, X³ is N—C(NH)—NH₂ and r is 1;-   X⁵ represents C(NH)—NH₂ or NH—C(NH)—NH₂, preferably C(NH)—NH₂;-   X⁶ represents CH or N.

Preferred compound of the invention are:

-   H—(R)Pro-Phe-Pab-   HOOC—CH₂—(R)Pro-Phe-Pab-   H—(R)Phe-Phe-Pab-   HOOC—CO—(R)Phe-Phe-Pab-   HOOC—CH₂—(R)Phe-Phe-Pab-   H—(R)Cha-Phe-Pab-   HOOC—CH₂—(R)Cha-Phe-Pab-   H—(R)Phe-Cha-Pab-   HOOC—CH₂—(R)Phe-Cha-Pab-   H—(R)Cha-Cha-Pab-   HOOC—CH₂—(R)Cha-Cha-Pab

Furthermore, it has been found that compounds of the general Formula V,either as such or in the form of physiologically acceptable salts, andincluding stereoisomers, are potent inhibitors of serine proteases,especially thrombin and kininogenases such as kallikrein after oral orparenteral administration:

wherein:

-   A¹ represents a structural fragment of Formula IIa, IIb, IIc, IId or    IIe;    wherein:-   k is an integer 0, 1, 2, 3 or 4;-   m is an integer 1, 2, 3 or 4;-   q is an integer 0, 1, 2 or 3;-   R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon    atoms and is possibly substituted in the position which is alpha to    the carbonyl group, and the alpha substituent is a group    R¹⁷—(CH₂)_(p)— wherein p is 0, 1 or 2 and R¹⁷ is COOR¹², CONHR¹²,    where R¹² is H or an alkyl group having 1 to 4 carbon atoms or a    benzyl group, and R¹¹ is H or an alkyl group having 1 to 6 carbon    atoms, or a benzyl group, or-   R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or-   R¹ represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4    carbon atoms and is possibly substituted alpha to the carbonyl with    an alkyl group having 1 to 4 carbon atoms and where R¹³ is H or an    alkyl group having 1 to 4 carbon atoms or —CH₂COOR¹²′ where R¹² is    as defined above, or-   R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to    4 carbon atoms and is possibly substituted alpha to the carbonyl    with an alkyl group having 1 to 4 carbon atoms and where R¹² is as    defined above, or-   R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,    Ph(2-COOR¹²)—SO₂—, where R¹² is as defined above and R¹⁴ is an alkyl    group having 1-4 carbon atoms, or-   R¹ represents —CO—R¹⁵ is wherein R¹⁵ is an alkyl group having 1-4    carbon atoms, or-   R¹ represents —CO—OR¹⁵, where R¹⁵ is as defined above, or-   R¹ represent —CO—(CH₂)—COOR¹², where R¹² is as defined above and p    is an interger 0, 1 or 2, or-   R² represents H or an alkyl group having 1 to 4 carbon atoms or    R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and,    where R²¹ is H, an alkyl group having 1 to 4 carbon atoms or a    benzyl group;-   R³ represents an alkyl group having 1-4 carbon atoms, and the alkyl    group may or may not carry one or more flourine atoms, or-   R³ represents a cyclopentyl, cyclohexyl- or a phenyl group which may    or may not be substituted with an alkyl group having 1 to 4 carbon    atoms, or-   R³ represents a phenyl group substituted with a OR³¹ group, where    R³¹ is H or an alkyl group having 1 to 4 carbon atoms and k is 0, 1,    or-   R³ represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or-   R³ represent a cis- or trans-decalin group and k is 0, 1, or-   R³ represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl which may or may    not be substituted with a OR³¹ group, where R³¹ is as defined above    and k is 0, 1, or-   R³ represents Si(Me)₃ or CH(R³²)₂₁ wherein R³² is a cyclohexyl- or a    phenyl group;-   R⁴ represents H, an alkyl group having 1 to 4 carbon atoms, a    cyclohexyl- or a phenyl group;-   A², B and n are defined as described under Formula I above;-   D is Z or (Z)₂1 wherein Z represents a benzyloxycarbonyl group.

The benzyloxycarbonyl group (Z or (Z)₂) will bind to the amidino- orguanidino nitrogens present in B.

Preferred and particularly preferred combinations are the same asdescribed for Formula I above.

Furthermore, it has been found that compounds of the general Formula Va,either as such or in the form of physiologically acceptable salts, andincluding stereoisomers, are potent inhibitors of thrombin after oral orparenteral administration:

wherein:

-   A¹ represents a structural fragment of Formula IIa, IIb, IIc or IId,    preferably IIa or IIb;    wherein:-   k is an integer 0, 1, 2, 3 or 4, preferably 0, 1;-   q is an integer 0, 1, 2 or 3, preferably 1;-   R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon    atoms and is possibly substituted in the position which is alpha to    the carbonyl group, and the alpha substituent is a group    R¹⁷-(CH₂)_(n)—, wherein p is 0, 1 or 2 and R¹⁷ is COOR¹², CONHR¹²;    where R¹² is H, an alkyl group having 1 to 4 carbon atoms or a    benzyl group, and R¹¹ is H or an alkyl group having 1 to 6 carbon    atoms, or a benzyl group, or-   R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or-   R¹ represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4    carbon atoms and is possibly substituted alpha to the carbonyl with    an alkyl group having 1 to 4 carbon atoms and where R¹³ is H or an    alkyl group having 1 to 4 carbon atoms or —CH₂COOR¹² where R¹² is as    defined above, or-   R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to    4 carbon atoms and is possibly substituted alpha to the carbonyl    with an alkyl group having 1 to 4 carbon atoms and where R¹² is as    defined above, or-   R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,    Ph(2-COOR¹²)—SO₂— where R¹² is as defined above and R¹⁴ is an    alkylgroup having 1-4 carbon atoms, or-   R¹ represents —CO—R¹⁵, wherein R's is an alkyl group having 1-4    carbon atoms, or-   R¹ represents —CO—OR¹⁵, where R¹⁵ is as defined above, or-   R¹ represent —CO—(CH₂)_(p)—COOR¹², where R¹² is as defined above and    p is an interger 0, 1 or 2, or    Preferably R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1    to 4 carbon atoms and R¹¹ is as defined above.-   R² represents H or an alkyl group having 1 to 4 carbon atoms, or    R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R²¹    is H or an alkyl group having 1 to 4 carbon atoms or a benzyl group;-   R³ represents an alkyl group having 1-4 carbon atoms, and the alkyl    group may or may not carry one or more fluorine atoms, or-   R³ represents a cyclopentyl, cyclohexyl- or a phenyl group which may    or may not be substituted with an alkyl group having 1 to 4 carbon    atoms, or-   R³ represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or-   R³ represent a cis- or trans-decalin group and k is 0, 1, or-   R³ represents Si(Me)₃ or CH(R³²)₂1 wherein R³² is a cyclohexyl- or    phenyl group;-   R⁴ represents an alkyl group having 1 to 4 carbon atoms, a    cyclohexyl or a phenyl group, preferably a cyclohexyl or a phenyl    group;-   A², B and n are defined as described under Formula Ia above;-   D is Z or (Z)₂;-   Z represents a benzyloxycarbonyl group.

Preferred integers, groups or combinations and particularly preferredcombinations are the same as described for Formula Ia above but R¹¹ isH, an alkyl group having 1 to 6 carbon atoms or a benzyl group.

Preferred compounds having Formula Va are:

-   BnOOC—CH₂—(R)Cgl-Aze-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pab (Z)-   BnOOC—CH₂—(R)Cgl-Pro-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pab (Z)-   BnOOC—CH₂)₂—(R)Cgl-Pro-Pab (Z)-   BnOOC—CH₂—(R,S)CH(COOBn)-(R)Cgl-Pic-Pab (Z)-   BnOOC—CH₂—(R)Cha-Aze-Pab(Z)-   BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Aze-Pab(Z)-   BnOOC—CH₂—(R or S)CH(COOBn)—(R)Cha-Aze-Pab(Z)/a-   BnOOC—CH₂—(R or S)CH(COOBn)—(R)Cha-Aze-Pab(Z)/b-   BnOOC—CH₂—CH₂—(R)Cha-Aze-Pab(Z)-   BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab(Z)-   BnOOC—CH₂—(R)Cha-Pro-Pab(Z)-   BnOOC—CH₂—(Me)(R)cha-Pro-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Cha-Pro-Pab(Z)-   BnOOC—CH₂—CH₂-(Me)(R)Cha-Pro-Pab(Z)-   BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pro-Pab(Z)-   BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab(Z)-   Ph(4-COOH)—SO₂—(R)Cha-Pro-Pab(Z)-   Boc-(R)Cha-Pic-Pab(Z)-   BnOOC—CH₂—(R)Cha-Pic-Pab(Z)-   BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pic-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Cha-Pic-Pab(Z)-   EtOOC—CO—(R)Cha-Pic-Pab(Z)-   MeOOC—CH₂—CO—(R)Cha-Pic-Pab(Z)-   H₂N—CO—CH₂—(R)Cha-Pic-Pab(Z)-   Ac—(R)Cha-Pic-Pab(Z)-   Me—SO₂—(R)Cha-Pic-Pab(Z)-   BnOOC—CH₂—(R)Cha-Val-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Cha-(R,S)Val-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Hoc-Aze-Pab(Z)-   BnOOC—CH₂—(R,S)CH(COOBn)-(R)Hoc-Pro-Pab (Z)-   BnOOC—CH₂—(R)Hoc-Pic-Pab(Z)-   (BnOOC—CH₂) 2-(R)Hoc-Pic-Pab (Z)-   BnOOC—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Tic-Pro-Pab(Z)-   BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pig(Z)₂-   BnOOC—CH₂—(R)Cgl-Pro-Pig(Z) 2-   BnOOC—CH₂—(R)Cgl-Aze-Pac(Z)-   BnOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab(Z)-   MeOOC—CH₂—(R)Cgl-Aze-Pab(Z)-   EtOOC—CH₂—(R)Cgl-Aze-Pab(Z)-   ^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab(Z)-   ^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab(Z)-   BnOOC—CH₂—(R)Cha-Pro-Pac(Z)-   BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pac(Z)-   BnOOC—CH₂—CH₂—(R)Cha-Aze-Pac(Z)-   BnOOC—CH₂—(R)Cha-Aze-Pig(Z)-   BnOOC—CH₂—(R)Cha-Pro-Pig(Z)-   BnOOC—CH₂—CH₂—(R)Cha-Pro-Pig(Z)-   (BnOOC—CH₂)₂—(R)Cgl-Pro-Pig(Z)-   BnOOC—CH₂—CH₂ (BnOOC—CH₂)—(R)Cha-Pro-Pig(Z)-   BnOOC—CH₂—(R)Cha-Pic-(R,S)Itp (Z)-   BnOOC—CH₂—(R)Cgl-Pro-(R,S)Hig(Z)-   BnOOC—CH₂—(R)Cgl-Aze-Rig(Z)-   BnOOC—CH₂—(R)Cha-Pro-Rig(Z)-   BnOOC—CH₂—CH₂—(R)Cha-Aze-Rig(Z)

Particularly preferred compounds are:

-   BnOOC—CH₂—(R)Cgl-Aze-Pab(z)-   BnOOC—CH₂—(R)Cha-Pro-Pab(Z)-   BnOOC—CH—(R)Cha-Pic-Pab(Z)-   BnOOC—CH₂—(R)Cgl-Pro-Pig(Z)₂-   EtOOC—CH₂—(R)Cgl-Aze-Pab(Z)-   BnOOC—CH₂—(R)Cha-Pro-Pac(Z)-   Bn OOC—CH₂—(R)Cha-Pro-Pig(Z)

Furthermore, it has been found that compounds of the general Formula Vb,either as such or in the form of physiologically acceptable salts, andincluding stereoisomers, are potent inhibitors of kallikrein after oralor parenteral administration:

wherein:

-   A¹ represents a structural fragment of formula IIa, IIb or IIe,    preferably IIa or IIb; wherein:-   k is an integer 0, 1, 2, 3 or 4, preferably 0, 1;-   q is an integer 0, 1, 2, or 3, preferably 1;-   R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon    atoms and is possibly substituted in the position which is alpha to    the carbonyl group, and the alpha substituent is a group    R¹⁷—(CH₂)_(n)—, wherein p is 0, 1 or 2 and R¹⁷ is COOR¹², CONHR¹²,    where R¹¹ is H or an alkyl group having 1 to 4 carbon atoms, and R¹¹    is H or an alkyl group having 1 to 6 carbon atoms, or a benzyl    group, or-   R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or-   R¹ represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4    carbon atoms and is possibly substituted alpha to the carbonyl with    an alkyl group having 1 to 4 carbon atoms and where R¹³ is H or an    alkyl group having 1 to 4 carbon atoms or —CH₂COOR¹² where R¹² is as    defined above, or-   R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to    4 carbon atoms and is possibly substituted alpha to the carbonyl    with an alkyl group having 1 to 4 carbon atoms and where R¹² is as    defined above, or-   R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂,    Ph(2-COOR¹²)—SO₂—, where R¹² is as defined above and R¹⁴ is an    alkylgroup having 1-4 carbon atoms, or-   R¹ represents —CO—R¹⁵, wherein R¹⁵ is an alkyl group having 1-4    carbon atoms, or-   R¹ represents —CO—OR¹⁵, where R¹⁵ is as defined above, or-   R¹ represent —CO—(CH₂)_(p)—COOR¹², where R¹² is as defined above and    p is 0, 1 or 2, or-   R² represents H or an alkyl group having 1 to 4 carbon atoms or    R²¹OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R²¹    is H, an alkyl group having 1 to 4 carbon atoms or a benzyl group;-   R³ represents an alkyl group having 1-4 carbon atoms, and the alkyl    group may or may not carry one or more fluorine atoms, or-   R³ represents a cyclopentyl, cyclohexyl- or a phenyl group which may    or may not be substituted with an alkyl group having 1 to 4 carbon    atoms, or-   R³ represents a phenyl group substituted with a OR³¹ group, where    R³¹ is H or an alkyl group having 1 to 4 carbon atoms and k is 0, 1,    or-   R³ represents a 1-naphthyl or 1-naphthyl group and k is 0, 1, or-   R³ represent a cis- or trans-decalin group and k is 0,1, or-   R³ represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl which may or may    not be substituted with a OR³¹ group, where R³¹ is as defined above    and k is 0, 1, or-   R³ represents Si(Me)₃ or CH(R³²)₂, wherein R³² is a cyclohexyl- or    phenyl group;-   R⁴ represents H, an alkyl group having 1 to carbon atoms, a    cyclohexyl or a phenyl group, preferably H;-   A², B and n are defined as described under Formula Ib above;-   D represents Z or (Z)₂.

Preferred integers, groups or combinations and particularly preferredcombinations are the same as described in Formula Ib above but R¹¹ is H,an alkyl group having 1 to 6 carbon atoms or a benzyl group.

Preferred compounds having Formula Vb are:

-   Boc-(R)Pro-Phe-Pab(Z)-   BnOOC—CH₂—(R)Pro-Phe-Pab(Z)-   Boc-(R)Phe-Phe-Pab(Z)-   MeOOC—CO—(R)Phe-Phe-Pab(Z)-   BnOOC—CH₂—(R)Phe-Phe-Pab(Z)

In a further embodiment the invention relates to novel use of a compoundof the formula:

as a starting material in synthesis of a peptidic serine proteaseinhibitor, and in particular in synthesis of peptidic thrombininhibitors or kininogenases inhibitors. It can be used as such or havingthe amidino group either mono- or diprotected at the nitrogens with aprotective group such as benzyloxy carbonyl. Protection of the amidinoderivatives is carried out by methods known in the art for amidinocompounds. This compound is named “1-amidino-4-aminomethylbenzene” or“H-Pab” herein. The compound has been previously disclosed in inter aliaBiochem. Pharm. vol 23, p. 2247-2256.

The structural fragment of the formula

has however not been previously disclosed as a structural element in apharmaceutically active compound, especially a peptic compound. Thefragment renders a serine protease inhibitor, and in particular athrombin inhibitor or kininogenases inhibitor valuable.

In a further embodiment the invention relates to novel use of a compoundof the formula:

as a starting material in synthesis of a thrombin inhibitor. Thecompound may have the amidino group either mono- or diprotected at thenitrogens with a protective group such as benzyloxy carbonyl. Protectionof the amidino derivatives is carried out by methods known in the artfor amidino compounds. This compound is named “1-amidino-4-aminomethylcyclohexane” or “H-Pac” herein.

The compound has been previously disclosed in DE 2748295.

The structural fragment of the formula

has however not been previously disclosed as a structural element in athrombin inhibitor valuable.

In a further embodiment the invention relates to a novel compound of theformula:

and the use of said compound as a starting material in synthesis of aserine protease inhibitor, especially a thrombin inhibitor orkininogenase inhibitor. The compound may have the amidino group eithermono- or diprotected at the nitrogens with a protective group such asbenzyloxy carbonyl. Protection of the amidino derivatives is carried outby methods known in the art for amidno compounds. This compound is named“4-aminoethyl-1-amidino piperidine” or “H—Rig” herein.

The structural fragment of the formula

has however not been previously disclosed as a structural element in apharmaceutically active compound, especially a peptic compound. Thefragment renders a serine protease inhibitor, and in particular athrombin inhibitor or kininogenases inhibitor varuable.

In a further embodiment the invention relates to a novel compound of theformula:

and the use of said compound as a starting material in synthesis of aserine protease inhibitor especially a thrombin inhibitor orkininogenase inhibitor. The compound may have the amidino group eithermono- or diprotected at the nitrogens with a protective group such asbenzyloxy carbonyl. Protection of the amidino derivatives is carried outby methods known in the art for amidino compounds. This compound isnamed “1,3-diaza-2-imino-4-aminoethyl cyclohexane” or “H-Itp” herein.

The structural fragment of the formula

has however not been previously disclosed as a strucural element in apharmaceutically active compound, especially a peptic compound. Thefragment renders a serine protease inhibitor, and in particular athrombin inhibitor or kiniogenases inhibitor varuable.

In a further embodiment the invention relates to novel compounds of theformula:

where n is 1 or 2

-   -   s is 0 ro 1,        and the use of said compounds as a starting material in        synthesis of serine protease inhibitors, especially thrombin        inhibitors or kininogenases inhibitors. The compound may have        the amidino group either mono- or diprotected at the nitrogens        with a protective group such as benzyloxy carbonyl. Protection        of the amidino derivatives is carried out by methods known in        the art for amidino compounds. These compounds are named:

-   1-amidino-3-aminomethylpyrrolidine or “H-Nig” when n is 1 and s is 1

-   1-amidino-3-aminoethyl pyrrolidine or “H-Hig” when n is 2 and s is 1

-   3-aminomethyl-1-amidino azetidine or “H-Mig” when n is 1 and s is 0

-   3-aminoethyl-1-amidino azetidine or “H-Dig” when n is 2 and s is 0

The structural fragment of the formula

has however not been previously disclosed as a structural element in apharmaceutically active compound, especially a peptic compound. Thefragment renders a serine protease inhibitor, and in particular athrombin inhibitor or kininogenases inhibitor valuable.

A further embodiment of the invention are the novel compounds having theamidino group mono- or di-protected at the nitrogens with a benzyloxycarbonyl group, examples of such compounds are

-   4-aminomethyl-1-(N-benzyloxycarbonylamidino)benzene (H-Pab(Z)),-   4-aminomethyl-1-(N,N′-di(benzyloxycarbonyl)amidino) benzene    (H-Pab(Z)₂),-   4-aminomethyl-1-(N-benzyloxycarbonylamidino) cyclohexane (H-Pac(Z)),-   4-aminomethyl-1-(N,N′-di(benzyloxycarbonyl)amidino) cyclohexane    (H-Pac(Z)₂),-   4-aminoethyl-1-(N-benzyloxy-carbonylamidino piperidine (H—Rig(Z)),-   4-aminoethyl-1-N,N′-di(benzyloxycarbonyl)amidino piperidine    (H—Rig(Z)₂),-   (3RS)-1-(N-benzyloxycarbonylamidino)-3-aminomethyl pyrrolidine    (H-Nig(Z)),-   (3RS)-1-(N,N′-di(benzyloxycarbonyl)amidino)-3-aminomethylpyrrolidine    (H-Nig(Z)₂),-   (3RS)-1-(N-benzyloxycarbonylamidino)-3-aminoethyl pyrrolidine    (H-Hig(Z)),-   (3RS)-1-(N,N′-di(benzyloxycarbonyl)amidino)-3-aminoethyl pyrrolidine    (H-Hig(Z)₂),-   3-aminomethyl-1-(N-benzyloxycarbonylamidino) azetidine (H-Mig(Z)),-   3-aminomethyl-1-(N,N′-di(benzyloxycarbonyl)amidino) azetidine    (H-Mig(Z)₂),-   3-aminoethyl-1-(N-benzyloxycarbonylamidino) azetidine (H-Dig(Z)),-   3-aminoethyl-1-(N,N′-di(benzyloxycarbonyl)amidino) azetidine    (H-Dig(Z)₂)

Said compounds are used as starting materials in the preparation of theclaimed peptide derivatives of formulas I, Ia, Ib, V, Va and Vb.

Medical and Pharmaceutical Use

The invention also provides compositions and methods for the treatment,in a human or animal organism, of conditions where inhibition ofthrombin is required and of physiologically disorders especiallyinflammatory diseases.

The thrombin inhibiting compounds of the invention are expected to beuseful in particular in animals including man in treatment orprophylaxis of thrombosis and hypercoagulability in blood and tissues.They are furthermore expected to be useful in situations where there isan undesirable excess of the thrombin without signs ofhypercoagulability, for example as in Alzheimers disease andpancreatitis. Disease states in which these compounds have a potentialutility, in treatment and/or prophylaxis, include venous thrombosis andpulmonary embolism, arterial thrombosis, such as in myocardialinfarction, unstable angina, thrombosis-based stroke and peripheralarterial thrombosis and systemic embolism usually from the atrium duringarterial fibrillation or from the left ventricule after transmuralmyocardial infarction. Further, these compounds have expected utility inprophylaxis of atherosclerotic diseases such as coronary arterialdisease, cerebral arterial disease and peripheral arterial disease.Further, these compounds are expected to have synergistic antithromboticeffects when combined with any antithrombotic agent with a differentmechanism of action, such as the antiplatelet agent acetylsalicylicacid. Further, these compounds are expected to be useful together withthrombolytics in thrombotic diseases, in particular myocardialinfarction. Further, these compounds have expected utility inprophylaxis for re-occlusion after thrombolysis, percutaneoustrans-luminal angioplasty (PTCA) and coronary bypass operations.Further, these compounds have expected utility in prevention ofre-thrombosis after microsurgery and vascular surgery in general.Further, these compounds have expected utility in treatment andprophylaxis of disseminated intravascular coagulation caused bybacteria, multiple trauma, intoxication or any other mechanism. Further,these compounds are expected to be useful in anticoagulant treatmentwhen blood is in contact with foreign surfaces in the body such asvascular grafts, vasculars stemts, vascular catheters, mechanical andbiological prosthetic or any other medical device. Further, thesecompounds have expected utility in anticoagulant treatment when blood isin contact with medical devices outside the body such as duringcardiovascular surgery using or heart-lung machine or in haemodialysis.

A further expected utility of the anticoagulant compounds of theinvention are in rinsing of catheters and mechanical devises used inpatients in vivo, and as anticoagulants for preservation of blood,plasma and other blood products in vitro.

The antiinflammatory inhibiting compounds of the invention are expectedto be useful in particular in animals including man in treatment orprophylaxis of inflammatory diseases such as asthma, rhinitis,pancreatitis, uticaria, inflammatory bowel diseases, and arthritis. Aneffective amount of kininogenase inhibiting compounds with or without aphysiologically acceptable carrier or diluent can be used solely or incombination with other therapeutic agents.

The compounds inhibit the activity of kallikreins assessed withchromogenic substrates according to known procedures. Theanti-inflammatory actions of the present compounds can for example bestudied by their inhibition of allergen-induced exudative inflammatoryprocesses in airway mucosa or gut mucosa.

Pharmaceutical Preparations

The compounds of the invention will normally be administered orally,rectally, dermally, nasally, tracheally, bronchially, parenterally orvia inhalation route, in the form of pharmaceutical preparationscomprising the active ingredient either as a free base or apharmaceutical acceptable non-toxic organic or inorganic acid additionsalt, e.g. the hydrochloride, hydrobromide, sulphate, hydrosulphate,nitrate, lactate, acetate, citrate, bensoate, succinate, tartrate,trifluoroacetate and the like in a pharmaceutically acceptable dosageform. Depending upon the disorder and patient to be treated and theroute of administration, the compositions may be administered at varyingdoses.

The dosage form may be a solid, semisolid or liquid preparation preparedby per se known techniques. Usually the active substance will constitutebetween 0.1 and 99% by weight of the preparation, more specificallybetween 0.1 and 50% by weight for preparations intended for parenteraladministration and between 0.2 and 75% by weight for preparationssuitable for oral administration.

Suitable daily doses of the compounds of the invention in therapeuticaltreatment of humans are about 0.001-100 mg/kg body weight at peroraladministration and 0.001-50 mg/kg body weight at parenteraladministration.

Preparation

A further objective of the invention is the mode of preparation of thecompounds. The compounds of Formula I and V may be prepared by processescomprise coupling of an N-terminally protected dipeptide or aminoacid,when a N-terminally amino acid is used a second aminoacid is addedafterwards using standard methods to a compoundH₂N—(CH₂)_(n)—Xwherein n is an integer 0, 1, 2, 3 or 4, X is B or B-D where B is asdefined in formula I and D is as defined in formula V as such or havingthe guanidino or amidino nitrogens either mono or diprotected with anamin protecting group such as a benzyloxy carbonyl-, tert-butyloxycarbonyl- or p-toluenesulphonyl-group or X is a group transferable intoB followed by removal of the protectary group(s) or deprotection of theN-terminal nitrogen followed by alkylation of the N-terminal nitrogenand if desired deprotection by known methods and if desired forming aphysiologically acceptable salt, and in those cases where the reactionresults in a mixture of stereoisomers, these are optionally separated bystandard chromatographic or re-crystallisation techniques, and ifdesired a single stereoisomer is isolated.

In more detail the compounds of Formula I or V may be prepared by eitherof the following methods:

Method Ia

Coupling of an N-terminally protected dipeptide, selected from A¹ and A²in Formulas I or V and prepared by standard peptide coupling, with acompound

using standard peptide coupling, shown in the formula

wherein n is as defined in Formula I W¹ is an N-teminal amino protectinggroup such as tert-butyloxy carbonyl and benzyloxy carbonyl and and Q¹is —C(NH)—NH₂, —C(NW²)—NH—W², —C(NH)—NH—W², —NH—C(NH)—NH₂,—NH—C(NH)—NH—W², —N(W²)—C(NH)—NH—W² or —NH—C(NW²)—NH—W², where W² is anamine protecting group such as tert-butyloxy carbonyl or benzyloxycarbonyl, or Q¹ is —CN, —CO—NH₂ or —CS—NH₂, where the group issubsequently transferred into a amidino group (e.g giving Q¹═—C(NH)—NH₂)by methods known in the art or Q¹ is NH₂ or NH—W², where W² is asdefined above, where the amino group is subsequently transferred into aguanidino group (giving Q¹═—NH—C(NH)—NH₂), after deprotection of theW²-group when Q¹ is —NH—W² (W² in this case must be orthogonal to W¹),by methods known in the art.

The final compounds can be made in any of the following ways, dependingon the nature of the Q¹-group used: Removal of the protecting group(s)(when Q¹=—C(NH)—NH₂, —C(NW²)—NH—W², —C(NH)—NH—W², —NH—C(NH)—NH₂,—NH—C(NH)—NH—W², —N(W²)—C(NH)—NH—W² or —NH—C(NW²)—NH—W²), or a selectivedeprotection of the W¹-group (e.g when Q¹=—C(NW²)—NH—W², —C(NH)—NH—W²,—NH—C(NH)—H—W², —N(W²)—C(NH)—NH—W² or —NH—C(NW²)—NH—W² (W² in this casemust be orthogonal to W¹) followed by alkylation of the N-terminalnitrogen by methods known in the art and if desired deprotection byknown methods.

Method Ib

Coupling of an N-terminally protected amino acid, selected from A² inFormulas I or V and prepared by standard methods, with a compound offormula

using standard peptide coupling, shown in the formula

wherein n, W¹, and Q¹ are as defined above followed by deprotection ofthe W¹-group and coupling with the N-terminal amino acid, in a protectedform, leading to the protected peptide described in Method Ia. Thesynthesis to the final peptides is then continued according to MethodIa.Method IIa

Coupling of an N-terminally protected dipeptide, selected from A¹ and A²in Formulas I or V and prepared by standard peptide coupling, with acompound

using standard peptide coupling, shown in the formula

wherein n is as defined in Formula I, W¹ is an N-teminal aminoprotecting group such as tertbutyloxy carbonyl and benzyloxy carbonyland and Q¹ is —C(NH)—NH₂, —C(NW²)—NH—W², —C(NH)—NH—W², —NH—C(NH)—NH₂,—NH—C(NH)—NH—W¹—N(W²)—C(NH)—NH—W² or —NH—C(W²)—NH—W² where W² is anamine protecting group such as tert-butyloxy carbonyl or benzyloxycarbonyl, or Q¹ is —CN, —CO—NH₂ or —CS—NH₂, where the group issubsequently transferred into a amidino group (e.g giving Q¹=—C(NH)—NH₂)by methods known in the art or Q¹ is NH₂ or NH—W², where W² is asdefined above, where the amino group is subsequently transferred into aguanidino group (giving Q¹═—NH—C(NH)—NH₂), after deprotection of theW²-group when Q¹ is —NH—W² (W² in this case must be orthogonal to W¹) bymethods known in the art.

The final compounds can be made in any of the following ways, dependingon the nature of the Q¹-group used: Removal of the protecting group(s)(when Q¹=—C(NH)—NH₂, —C(NW²)—NH—W², —C(NH)—NH—W², —NH—C(NH)—NH₂,—NH—C(NH)—NH—W², —N(W²)—C(NH)—NH—W² or —NH—C(NW²)—NH—W²), or a selectivedeprotection of the W¹-group (e.g when Q¹═—C(NW²)—NH—W², —C(NH)—NH—W²,—NH—C(NH)—NH—W², —N(W²)—C(NH)—NH—W² or —NH—C(NW²)—NH—W² (W² in this casemust be orthogonal to W¹) followed by alkylation of the N-terminalnitrogen by methods known in the art and if desired deprotection byknown methods.

Method IIb

Coupling of an N-terminally protected amino acid, =elected from A² inFormulas I or V and prepared by standard methods, with a compound offormula

using standard peptide coupling, shown in the formula

wherein n, W¹ and Q¹ are as defined above followed by deprotection ofthe W¹-group and coupling with the N-terminal amino acid, in a protectedform, leading to the protected peptide described in Method IIa. Thesynthesis to the final peptides is then continued according to MethodIIa.Method IIIa

Coupling of an N-terminally protected dipeptide, selected from A¹ and A²in Formulas I or V and prepared by standard peptide coupling, with acompound

using standard peptide coupling, shown in the formula

wherein n is as defined in Formula I and r is 0.1 when X¹, X² and X⁴ areCH₂ or r is 0 when X² and X⁴ are CH₂ and X¹ is abscent, W¹ is anN-teminal amino protecting group such as tert-butyloxy carbonyl andbenzyloxy carbonyl and and Q² is —C(NH)—NH₂, —C(NW²)—NH—W², or—C(NH)—NH—W², where W² is an amine protecting group such astert-butyloxy carbonyl or benzyloxy carbonyl, or Q² is equal to W² wherethe amino group, after deprotection of the W² group (W² in this casemust be orthogonal to W¹), is subsequently transferred into a guanidinogroup using a unprotected, N-protected or N,N′-diprotected guanidationreagent by methods known in the art (giving Q²═—C(NH)—NH₂, —C(NW²)—NH—W²or —C(NH)—NH—W²).

The final compounds can be made in any of the following ways, dependingon the nature of the Q²-group used: Removal of the protecting group(s)(when Q²═—C(NH)—NH₂, —C(NW²)—NH—W² or —C(NH)—NH—W²), or a selectivedeprotection of the W¹-group (e.g when Q²═—C(NW²)—NH—W², —C(NH)—NH—W²,W² in this case must be orthogonal to W¹) followed by alkylation of theN-terminal nitrogen by methods known in the art and if desireddeprotection known methods.

Method IIIb

Coupling of an N-terminally protected amino acid, selected from A² inFormulas I or V and prepared by standard methods, with a compound offormula

using standard peptide coupling, shown in the formula

wherein n, r, X¹, X² and X⁴ W¹, and Q² are as defined above followed bydeprotection of the W¹-group and coupling with the N-terminal aminoacid, in a protected form, leading to the protected peptide described inMethod IIIa. The synthesis to the final peptides is then continuedaccording to Method IIIa.Method IVa

Coupling of an N-terminally protected dipeptide, selected from A¹ and A²in Formulas I or V and prepared by standard peptide coupling, with acompound

using standard peptide coupling, shown in the formula

wherein n is as defined in Formula I, W¹ is an N-terminal aminoprotecting group such as tert-butyloxy carbonyl or benzyloxy carbonyland W³ is H or an amino protecting group such as aryl sulfonyl,benzyloxy carbonyl or tert-butyloxy carbonyl. The final compounds can bemade in any of the following ways: Removal of the protecting group(s),or a selective deprotection of the W¹-group (W¹ must be orthogonal toW³) followed by alkylation of the N-terminal nitrogen and if desireddeprotection.Method IVb

Coupling of an N-terminally protected amino acid, selected from A² inFormulas I or V and prepared by standard methods, with a compound offormula

using standard peptide coupling, shown in the formula

wherein n, W¹, and W³ are as defined above followed by deprotection ofthe W¹-group (W¹ must be orthogonal to W³) and coupoing with theN-terminal amino acid, in a protected form, leading to the protectedpeptide described in Method IVa. The synthesis to the final peptides isthen continued according to Method IVa.

DETAILED DESCRIPTION OF THE INVENTION

The following description is illustrative of aspects of the invention.

Experimental Part

General experimental Procedures.

Mass spectra were recorded on a Finnigan MAT TSQ 700 triple quadropolemass spectrometer equipped with an electrospray interface.

The ¹H NMR and ¹³C NMR measurements were performed on BRUKER AC-P 300and BRUKER AM 500 spectrometers, the former operating at a ¹H frequencyof 500.14 MHz and a ¹³C frequency of 125.76 MHz and the latter at ¹H and¹³C frequency of 300.13 MHz and 75.46 MHz respectively.

The samples were about 10-50 mg dissolved in 0.6 ml of either of thefollowing solvents; CDCl₃ (isotopic purity>99.8%), CD₃OD (isotopicpurity>99.95%), D₂O (isotopic purity>99.98%) or DMSO-d₆ (isotopicpurity>99.8%). All solvents where purchased from Dr. Glaser A G, Basel.

The ¹H and ¹³C chemical shift values in CDCl₃ and CD₃OD are relative totetramethylsilane as an external standard. The ¹H chemical shifts in D₂Oare relative to the sodium salt of 3-(trimethylsilyl)-d₄-propanoic acidand the ¹³C chemical shifts in D₂O are referenced relative to1,4-dioxane (67.3 ppm), both as external standard. Calibrating with anexternal standard may in some cases cause minor shift differencescompared to an internal standard, however, the difference in ¹H chemicalshift is less than 0.02 ppm and in ¹³C less than 0.1 ppm.

The ¹H NMR spectrum of peptide sequences containing a proline or a“proline like” residue frequently exhibits two sets of resonances. Thiscorresponds to the existence of two contributing conformers with respectto the rotation around the amide bond, where proline is the N-part ofthe amide bond. The conformers are named cis and trans. In our compoundsthe sequences (R)Cha-Aze-, (R)Cha-Pro- and (R)Cha-Pic- often give riseto a cis-trans equilibrium with one conformer as the preponderantconformer (>90%). In those cases only the ¹H chemical shifts of themajor rotamer is reported. Only in the cases where the signals of theminor rotamer are clearly resolved they are reported in the NMRdocumentation. The same criterium is valid for the NH-signals in CDCl₃,only in the cases where the signals are clearly resolved they arereported in the NMR-documentation. This implies that the number ofprotons reported for some of the intermediates are less than the numberof protons expected from the chemical formula.

Thin-Layer Chromatography was carried out on commercial Merck Silicagel60F₂₅₄ coated glass or aluminium plates. Visualisation was by acombination of UV-light, followed by spraying with a solution preparedby mixing 372 ml of EtOH(95%), 13.8 ml of concentrated H₂SO₄, 4.2 ml ofconcentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde orphosphomolybdic acid reagent (5-10 w.t % in EtOH(95%)) and heating.

Flash chromatography was carried out on Merck Silica gel 60 (40-63 mm,230-400 mesh) under pressure of air.

Reversed phase high-performance liquid chromatography (in the Examplesreferred to as RPLC) was performed on a Waters M-590 instrument equippedwith three reverse phase Kromasil 100,C8 columns (Eka-Nobel) havingdifferent dimensions for analytical (4.6 mm×250 mm), semipreparative(1″×250 mm) and preparative (2″×500 mm) chromatography detecting at 226nm.

Freeze-drying was done on a Leybold-Heraeus, model Lyovac GT 2,apparatus.

Preparation of Starting Materials

Boc-(R)Pgl-OH

Prepared in the same way as described for Boc-(R)Cha-OH (vide infra)from H—(R)Pgl-OH.

Boc-(R)Cha-OH

To a solution of H—(R)Cha-OH, 21.55 g (125.8 mmol), in 130 ml 1 M NaOHand 65 ml THF was added 30 g (137.5 mmol) of (Boc)₂O and the mixture wasstirred for 4.5 h at room temperature. The THF was evaporated and anadditional 150 ml of water was added. The alkaline aqueous phase waswashed twice with EtOAc, then acidified with 2 M KHSO₄ and extractedwith 3×150 ml of EtOAc. The combined organic phase was washed withwater, brine and dried (Na₂SO₄). Evaporation of the solvent afforded30.9 g (90.5%) of the title compound as a white solid.

Boc-(R)Hop-OH

Prepared by the same procedure as described for Boc-(R)Cha-OH startingfrom H—(R)Hop-OH.

¹H-NMR (300 MHz, CDCl₃): δ 1.45 (s, 9H), 2.00 (m, 1H), 2.22 (m, 1H),2.75 (bt, 2H), 4.36 (bs, 1H), 5.05 (bs, 1H), 7.15-7.33 (m, 5H).

4-(tert-butyloxycarbonylaminomethyl)pyridin

To a solution of 10.81 g (100 mmol) 4-aminomethyl pyridine in 100 ml THFwas added 24 g (110 mmol) Boc₂O dissolved in 70 ml THF at 10° C. for 20minutes. The solution was allowed to reach room temperature and stirredfor 4 h (a precipitate was formed during the reaction and the slurrybecame red). The solvent was removed and the residue was dissolved inEtOAc and filtered through silica gel. Evaporation of the solvent gavethe title compound as a red oil which crystallized on standing. Thecrude product was used without further purification.

¹H-NMR (300 MHz, CDCl₃): δ 1.45 (s, 9H), 4.32 (d, 2H), 5.05 (bs,1H(NH)), 7.2 (d, 2H), 8.55 (d, 2H).

4-aminomethyl-1-(N-benzyloxycarbonylamidino)-benzene (H-Pab(Z)) (i)4-cyanobenzyl azide

A solution of 20.23 g (0.31 mol) sodium azide in 50 ml water was addedto 49.15 g (251 mmol) 4-cyanobenzyl bromide in 200 ml DMF at ambienttemperature. An exothermic reaction took place and after 1.5 h thereaction mixture was diluted with 200 ml toluene (caution: In order toavoid separation of potentially explosive azide compounds it isadviceable to add the toluene to the rection mixture before addition ofthe water) and 500 ml water. The aqueous phase was extracted with anadditional 2×50 ml toluene. The combined organic extracts were washedwith 2×50 ml water and brine and finally dried (MgSO₄) and filtered. Thesolution was used as such in the next step.

¹H-NMR (300 MHz, CDCl₃); δ 4.4 (s, 2H), 7.4 (d, 2H), 7.7 (d, 2H)

(ii) 4-amidino benzyl azide

Hydrogen chloride was bubbled into a mixture of 250 ml absolute ethanoland the solution from step (i) (approximatly 200 ml) above at −5° C.until saturation. Storage at 8° C. for 24 h and evaporation of most ofthe solvent followed by precipitation by addition of anhydrous ethergave white crystals which were isolated by filtration and dissolved in1.8 l of alcoholic ammonia. After 48 h most of the solvent was removedand 200 ml 3.75 M NaOH solution was added whereupon 4-amidino benzylazide precipitated as colourless crystals. The crystals were isolated byfiltration. At this point the yield of 4-amidino benzyl azide was 22.5 g(total 51%).

Ethylimidatobenzyl azide hydrochloride:

¹H-NMR (500 MHz, CD₃OD); δ 1.6 (t, 3H), 4.5 (s, 2H), 4.65 (q, 2H), 4.8(br s, 2H), 7.6 (d, 2H), 8.1 (d, 2H)

4-amidino benzyl azide:

¹H-NMR (500 MHz, CDCl₃); δ 4.3 (s, 2H), 5.7 (br s, 3H), 7.3 (d, 2H), 7.6(d, 2H).

¹³C-NMR (125 MHz, CDCl₃): amidine carbon: δ 165.5.

(iii) 4-(benzyloxycarbonylamidino)benzyl azide

The crystals from (ii) above were dissolved in 500 ml methylene chlorideand the resulting solution was dried (K₂CO₃), filtered and 27 ml (194mmol) triethyl amine was added. 25 ml Benzyl chloroformate was slowlyadded to the stirred solution while the reaction mixture was cooled inan ice bath. After 30 minutes an additional 2 ml benzyl chloroformatewas added and stirring was continued for another 30 minutes.Subsequently, water was added and the aqueous phase was adjusted to pH 7with 2M HCl. The organic phase was dried (MgSO₄) and the solvent wasremoved in vacuo. 4-(benzyloxycarbonylamidino)benzyl azide was finallyisolated as colorless crystals from ether/methylene chloride/hexane.

¹H-NMR (500 MHz, CDCl₃); δ 4.4 (s, 2H), 5.3 (s, 2H), 6.3-7.0 (br s, 1H),7.3-7.4 (m, 5H), 7.5 (d, 2H), 7.9 (d, 2H), 9.3-9.6 (br s, 1H).

¹³C-NMR (125 MHz, CDCl₃): amidine carbon: δ 167.5.

(iv) 4-aminomethyl-1-(N-benzyloxycarbonylamidino)-benzene (H-Pab(Z))

26.3 g (100 mmol) triphenylphosphine was added at room temperature tothe 4-(benzyloxycarbonylamidino)benzyl azide from (iii) above dissolvedin 160 ml THF. After 16 h an additional 6.6 g (25 mmol)triphenylphosphine was added and the solution was allowed to stand for 4h before removal of the solvent in vacuo. The residue was dissolved inmethylene chloride and extracted with 2M HCl. The aqueous phase waswashed with methylene chloride and ether and was subsequently madealcaline with 3.75M sodium hydroxide solution. Extraction with methylenechloride followed by drying (K₂CO₃) and removal of the solvent in vacuogave 20 g (The total yield starting from cyanobenzyl bromide is 28%) ofa yellow oil which solidified on standing.

¹H-NMR (500 MHz, CDCl₃); δ 1.2-2.2 (br s, 2H), 3.8 (s, 2H), 5.2 (s, 2H),7.2-7.35 (m, 5H), 7.4 (d, 2H), 7.8 (d, 2H), 9.1-9.6 (br s, 1H).

¹³C-NMR (125 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.6 and168.17.

H-Pig(Z)₂

(i) 4-(tert-butyloxycarbonyl-aminomethyl)piperidine

To a solution of 17.7 g 4-tert-butyloxycarbonylaminomethyl pyridine in125 ml MeOH was added 2 g of 5% Rh/Al₂03 and the mixture washydrogenated at 0.34 MPa over night. ¹H-NMR showed that thehydrogenation was incomplete. Therefore, the catalyst was filtered offand the solvent removed in vaccuo and the residue was dissolved in 100ml acetic acid, 2 g of 5% Rh/Al₂O₃ was added and the mixture washydrogenated for 4 days at 0.34 MPa. The catalyst was filtered off andmost of the acetic acid was removed in vaccuo. After addition of 50 mlwater to the residue the mixture was made alkaline with 5 M NaOH and thewater phase was extracted with 1×200+1×100 ml CH₂Cl₂. The combinedorganic phase was washed with 25 ml water and dried (MgSO₄). Evaporationof the solvent gave a 17.2 g of a brownish oil which was dissolved in 50ml of diethyl ether. Addition of 200 ml pentane gave a precipitate whichwas filtered off to give 7.7 g of a brown powder. Evaporation of themother liqour gave 7 g of a white oil. The brown powder was dissolved in100 ml EtOAc and the organic phase was washed with 1×50 ml+1×25 ml 1 MKHSO₄ The combined acidic phase was made alkaline with 2 M NaOH andextracted with 1×200+1×75 ml EtOAc. The combined organic phase was driedand evaporated to give 5.2 g of the title compound as a white powder.

Treatment of the white oil obtained from the mother liqour above in thesame way afforded an additional 3.4 g of the product. Total yield 40%.

¹H-NMR (500 MHz, CDCL₃, mixture of two rotamers, 3:1): major rotamer: δ1.11 (dq, 2H), 1.44 (s, 9H), 1.49-1.60 (m, 1H), 1.63-1.70 (m, 2H), 2.58(dt, 2H), 2.93-3.03 (m, 2H), 3.07 (m, 2H), 4.75 (bs, 1H(NH)).

Resolved signals arising from the minor rotamer appear at δ 1.21 (dq)and 1.91 (dt).

(ii) Boc-Pig(Z)₂

To a solution of 2 g (9.33 mmol) 4-(tert-butyloxycarbonyl-aminomethyl)piperidine in 60 ml CH₃CN was added 3.34 g (9.33 mmol) ofN,N′-(dibenzyloxy-carbonyl)methylisothiourea and the mixture was stirredat 60° C. for 22 h. The solvent was evaporated and the residue wasdissolved in EtOAc. The organic phase was washed with 2×20 ml 1 M KHSO₄₁1×20 ml water, 1×20 ml brine and dried (MgSO₄). Evaporation of thesolvent followed by flash chromatography using pethroleum ether/EtOAc(1/1) as eluent afforded 2.43 g (50%) of the desired product.

¹H-NMR (500 MHz, CDCl₃): Some signals, especially in the piperidinering, are selectively broadend due to an intramolecular exchangeprocess. This is especially pronounced for the 2- and 6-CH₂ groups ofthe piperidine ring, which exhibit a broad peak ranging from 3.7 to 4.5ppm.

δ 1.19-1.31 (m, 2H), 1.43 (s, 9H), 1.63-1.80 (m, 3H), 2.66-3.05 (m, 4H),3.7-4.5 (bs, 2H), 4.65 (bt, 1H(NH)), 5.13 (s, 4H), 7.2-7.4 (m, 10H),10.5 (bs, 1H(NH)).

(iii) H-Pig(Z)₂

A solution of 163 mg (0.31 mmol) Boc-Pig(Z)₂ in 5 ml EtOAc saturatedwith HCl(g) was stirred at ambient temperatur for 3 h and 20 minutes.The solvent was evaporated and the residue was dissolved in 30 mlCH₂Cl₂. The organic phase was washed with 5 ml 2 M NaOH, 1×5 ml water,1×5 ml brine and dried (MgSO₄). Evaporation of the solvent afforded 100mg (76%) of the title compound.

¹H-NMR (500 MHz, CDCl₃): Some signals, especially in the piperidin ring,are selectively broadend due to an intramolecular exchange process. Thisis especially pronounced for the 2- and 6-CH₂ groups of the piperidinering, which exhibit a broad peak ranging from 3.7 to 4.5 ppm.

δ 1.18-1.37 (m, 2H), 1.46-1.63 (m, 1H), 1.68-1.83 (m, 2H), 2.57 (d, 2H),2.86-3.03 (m, 2H), 3.7-4.5 (bs, 2H), 5.13 (s, 4H), 7.2-7.4 (m, 10H).

4-aminomethyl-1-(N-benzyloxy carbonylamidino)-cyclohexane(H-Pac(Z)×2HCl) (i) N-[N-4-(benzyloxycarbonyl)amidino benzyl]tert-butylcarbamate

1.466 g (6.7 mmol) (Boc)₂O was added to a stirred ice cold solution of1.81 g (6.4 mmol) 4-(benzyloxycarbonyl)amidino benzyl amine and 1 ml(7.1 mmol) triethyl amine in 25 ml methylene chloride. After 20 minutesmore methylene chloride was added and the mixture was washed with 5%acetic acid and 10% sodium carbonate solution. Drying (magnesiumsulphate) and removal of the solvent in vauo left a residue which couldbe crystallised from methylene chloride/hexane. The yield was 1.66 g(68%).

(ii) N-[N-4-amidino benzyl]tert-butyl carbamate

A mixture of 1.60 g (4.2 mmol) N-[4-(benzyloxycarbonyl)amidinobenzyl]tert-butyl carbamate, 5 ml acetic acid, and 160 mg 10% palladiumon charcoal in 50 ml ethanol was sirred in an atmosphere of hydrogen for2 h. The catalyst was removed by filtration through celite and thesolvent was removed in vacuo to give the acetate of the title compoundin quantitative yield.

(iii) N-[4-amidino cyclohexyl methyl]tert-butyl carbamate

17 mmol of the acetate of N-[4-amidino benzyl]tert-butyl carbamate washydrogenated in 100 ml metanol in the presence of 863 mg 5% rhodium onalumina at 3.4 MPa for 20 h. The catalyst was removed by filtration andthe solvent was removed in vacuo. The residue was dissolved in water andthe solution was made basic with sodium hydroxide. Subsequent extractionwith methylene chloride, drying of the combined organic phases(potassium carbonate) and removal of the solvent in vacuo gave 3.8 g(87%) of the title compound.

(iv) N-[N-4-(benzyloxycarbonyl)amidino cyclohexyl methyl]tert-butylcarbamate

1.25 ml (8.8 mmol)benzyl chloroformate was added at 0° C. to a stirredsolution of 2.04 g (8 mmol) N-[4-amidino cyclohexyl]tert-butylcarbamate, 1.23 ml (8.8 mmol) triethyl amine, and 197 mg DMAP in 40 mlmethylene chloride. After 10 minutes the reaction mixture was dilutedwith methylene chloride and extracted with water, dilute acetic acid,and sodium hydrogen carbonate solution. The organic phase was applied ona column of silica and subsequent elution with methylene chloridecontaining increasing amounts of ethyl acetate yielded 2.49 g (80%) ofthe title compound.

(v) 4-aminomethyl-1-(N-benzyloxy carbonylamidino)-cyclohexane(H-Pac(Z)×2HCl)

Hydrogen chloride was passed through a solution of 2 g (5.1 mmol)N-[4-(benzyloxycarbonyl)amidino cyclohexyl methyl]tert-butyl carbamatein 40 ml ethyl acetate. After 10 minutes methanol was added and uponremoval of some of the solvent in vacuo the dihydrochloride of titlecompound crystallised.

4-aminomethyl-1-(N-benzyloxy carbonylamidino) piperidine (H-Pig(Z)×HCl)(i) 4-(N-tert-butyloxycarbonylaminomethyl)-1-(N-benzyloxycarbonylamidino)piperidine (Boc-Pig(Z))

7.8 g (36.4 mmole) of 4-(N-tert-butyloxycarbonylaminomethyl) piperidineand 8.98 g (40 mmole) of N-benzyloxycarbonyl-S-methylisothiourea wasmixed in 25 mL ethanol. The mixture was heated at 60-70° C. for sixhours and left at roomtemperature for two days. The solvent wasevaporated and the residue was dissolved in CH₂Cl₂. The organic layerwas washed twice with 0.3 M KHSO₄ and once with brine. The combinedorganic layer was dried (Na₂SO₄), filtered and evaporated. The crudeproduct was purified by flash chromatography using a stepwise gradientof CH₂Cl₂/MeOH (100/0, 97/3, 95/5, 90/10) as eluent to yield 5.22 g(37%) of the title product.

(ii) H-Pig(Z)×HCl (4-aminomethyl-1-(N-benzyloxycarbonylamidino)piperidine

5.22 g (13.5 mmole) of Boc-Pig(Z) was dissolved in 100 mL ethyl acetatesaturated with HCl(g). The mixture was allowed to stand for one hour andthen evaporated. The residue was dissolved in water and washed with amixture of diethylether and ethyl acetate. The water layer wasfreeze-dried to yield 4.0 g (91%) of the title compound.

¹H-NMR (D₂O, 300 MHz): δ 1.40-1.60 (m, 2H), 2.05 (bd, 2H), 2.19 (m, 1H),3.07 (d, 2H), 3.34 (bt, 2H), 4.08 (bd, 2H), 5.40 (s, 2H), 7.5-7.63 (m,5H)

MS m/z 291 (M⁺+1)

4-Aminoethyl-1-benzyloxycarbonylamidino piperidine (H—Rig(Z)) (i)1-Benzyloxycarbonylamidino-4-hydroxyethyl piperidine

A mixture of 6.2 g (0.028 mol) of 4-hydroxyethyl piperidine and 3.6 g(0.028 mol) of N-benzyloxycarbonyl-S-methyl isothiourea in 50 ml ofacetonitrile was refluxed overnight. Evaporation and flashchromatography on silica gel with ethyl acetate gave 3.5 g (41%) of thetitle compound.

¹H-NMR (300 MHz, CDCl₃): δ 1.1-1.85 (m, 7H), 2.83 (bt, 2H), 4.70 (bt,2H), 4.18 (bd, 2H), 5.12 (s, 2H), 6.9-7.2 (m, 2H), 7.2-7.5 (m, 5H).

(ii) 1-Benzyloxycarbonylamidino-4-mesyloxyethyl piperidine

To an ice cooled solution of 3.50 g (0.0115 mol) of1-benzyloxy-carbonylamidino-4-hydroxyethyl piperidine, 1.15 g (0.0115mol) of triethyl amine in 40 ml of methylene chloride and 10 ml oftetrahydrofurane was added dropwise 1.30 g (0.115 mol) of mesylchloride.

The reaction mixture was allowed to stir for 1 h. The mixture was pouredinto water and the organic layer was kept. The aqueous layer wasextracted with methylene chloride and the combined organic layers werewashed with water, dried (Na₂SO₄) and evaporated. The product was usedwithout further purification in the next step. Yield: 4.4 q (100%).

¹H NMR (500 MHz, CDCl₃) d 1.15-1.3 (m, 2H), 1.65-1.8 (m, 5H), 2.84 (bt,2H), 3.01 (s, 3H), 4.20 (bd, 2H), 4.27 (t, 2H), 5.12 (s, 2H), 7.1-7.5(m, 7H).

(iii) 4-Azidoethyl-1-benzyloxycarbonylamidino piperidine

In 100 ml of dimethylformamide was dissolved 4.4 g (0.0115 mol) of crude1-benzyloxycarbonylamidino-4-mesyloxyethyl piperidine and 4.5 g (0.069Mol) of sodium azide was added. The mixture was heated at 100° C. for2.5 h. It was then poured into water and extracted with ethyl acetatethree times. The combined organic phase was washed with water, dried(Na₂SO₄) and evaporated. The residue was flash chromatographed on silicagel using ethyl acetate/heptane 1/1 as eluent. Yield: 3.0 g (79%).

¹H-NMR (500 MHz, CDCl₃) δ 1.20 (dq, 2H), 1.5-1.8 (m, 5H), 2.85 (dt, 2H),3.35 (t, 2H), 4.22 (bd, 2H), 5.13 (s, 2H), 6.9-7.2 (b, 2H), 7.2-7.45 (m,5H).

(iv) 4-Aminoethyl-1-benzyloxycarbonylamidino piperidine (H—Rig(Z))

To 30 ml of water was added 0.40 g of 10% Pd/C. Sodium borohydride, 1.0g (0.031 mol), was dissolved in 30 ml of water and was added carefullyto the stirred and ice-cooled slurry of Pd/C and water.4-Azidoethyl-1-benzyloxycarbonylamidino piperidine, 2.9 g (8.8 mmol),was dissolved in 80 ml of tetrahydrofurane and this solution was addeddropwise to the ice-cooled aqueous slurry above. After 4 h of stirringat room temperature the mixture was ice-cooled again and 30 ml of 2 MHCl was added. The mixture was filtered through celite and the celitewas rinsed with additional water. The tetrahydrofuran was evaporated andthe aqueous phase was washed with ethyl acetate. The aqueous phase wasmade alkaline with 2 M NaOH and extracted with methylene chloride threetimes. The combined organic phase was washed with water, dried (Na₂SO₄)and evaporated. The product was used in the following step withoutfurther purification.

¹H-NMR (500 MHz, CDCl₃) δ 1.1-1.5 (m, 6H), 1.55-1.65 (m, 1H), 1.73 (bd,2H), 2.72 (b, 2H), 2.81 (dt, 2H), 4.20 (bd, 2H), 5.12 (s, 2H), 6.9-7.2(b, 2H), 7.2-7.5 (m, 5H).

(3RS)-1-(N-benzyloxycarbonylamidino)-3-aminomethylpyrrolidine(H—(R,S)Nig(z)) (i) (3RS)-3-hydroxymethylpyrrolidine

16.4 g (0.0857 mole) (3RS)-1-benzyl-3-hydroxymethyl pyrrolidine (SeeH—(R,S)Hig(Z) (i) vide supra) was mixed with 1.6 g Pd/C (10%), 5 mlwater and 150 ml ethanol and the mixture was hydrogenated at 0.26 MPaover night. After filtration through hyflo and evaporation of thesolvent the ¹H-NMR showed that the reaction was not completed. Continuedhydrogenation at 0.26 MPa over 1.6 g Pd/C (10%) in 5 ml water/150 mlethanol for three days completed the reduction. Filtration through hyfloand evaporation of the solvent gave the product in a quantitative yield.

(ii) (3RS)-1-(N-benzyloxycarbonylamidino)-3-hydroxymethylpyrrolidine

1.01 g (0.01 mole) (3RS)-3-hydroxymethylpyrrolidine and 2.29 g (0.011mole) N-benzyloxycarbonyl-O-methylisourea was dissolved (the amine notvery soluble) in toluene and heated to 60° C. for three hours followedby stirring at room temperature over night. The mixture was evaporatedand the ¹H-NMR showed that the reaction was not completed. The mixturewas therefore dissolved in 15 ml acetonitrile and heated to 60° C. forthree hours followed by stirring at room temperature over night. Thesolvebt was evaporated and the mixture was dissolved in CH₂Cl₂, washedonce with water, dried (Na₂SO₄), filtered and evaporated. The crudeproduct was purified by flash chromatography using CH₂Cl₂/MeOH 95/5 aseluent to yield 0.70 g (25%) of the product.

MS m/z 278 (M⁺+1)

(iii) (3RS)-1-(N-benzyloxycarbonylamidino)-3-mesyloxymethyl pyrrolidine

0.7 g (2.53 mmole)(3RS)-1-(N-benzyloxycarbonylamidino)-3-hydroxymethylpyrrolidine and 0.70ml (5.05 mmole) triethylamine was dissolved in 15 ml diethylether/CH₂Cl₂ 1/1 and the mixture was cooled to 0° C. 0.25 ml (3.29 mole)methanesulphonyl chloride in 3 ml diethyl ether was slowly added and thereaction mixture was stirred at 0° C. for three hours. The solvent wasevaporated and the residue was dissolved in ethyl acetate and extractedwith a 0.3 M KHSO₄-solution. The water layer was washed once with CH₂Cl₂The water layer was made neutral with 10M NaOH-solution and extractedtwice with CH₂Cl₂. The combined organic layer was dried (Na₂SO₄),filtered and evaporated to yield 0.450 g (50%) of the title compound.

(iv) (3RS)-1-(N-benzyloxycarbonylamidino)-3-azidomethyl pyrrolidine

0.450 g (1.27 mmole)(3RS)-1-(N-benzyloxycarbonylamidino)-3-mesyloxymethyl pyrrolidine and0.124 g (1.9 mmole) of sodium azide were dissolved in 10 mldimethylformamide and heated to 60° C. for four hours followed bystirring at room temperature over night. Water was added and the mixturewas extracted twice with toluene/ethyl acetate 2/1. The combined organiclayer was dried (Na₂SO₄), filtered and evaporated. The crude product waspurified by flash chromatography using CH₂Cl₂/MeOH 95/5 as eluent toyield 0.262 g (68%) of the product.

MS m/z 303 (M⁺+1)

(v) (3RS)-1-(N-benzyloxycarbonylamidino)-3-aminomethyl pyrrolidine(H—(R,S)Nig(Z))

32 mg Pd/C (10%) and 2.6 ml H₂O was mixed and a gentle stream ofnitrogen was passed. 98 mg NaBH₄ in 2.6 ml H₂O was added folowed by aslow addition of 262 mg (0.87 mmole)(3RS)-1-(N-benzyloxycarbonylamidino)-3-mesyloxymethyl pyrrolidinedissolved in 7 ml MeOH. The mixture was allowed to stand for one hour. 5ml 1M HCl was added and the mixture was filtered through hyflo. Theorganic solvent was evaporated under reduced pressurea and the remainingwater layer was washed once with ethyl acetate, made alkaline withNaOH-solution and extracted several times with CH₂Cl₂. The combinedorganic layer was dried (Na₂SO₄), filtered and evaporated to yield 130mg (54%) of the product.

MS M/z 277 (M⁺+1)

(3RS)-1-(N-benzyloxycarbonylamidino)-3-aminoethyl pyrrolidine(H—(R,S)Hig(z)) (i) (3RS)-1-benzyl-3-hydroxymethylpyrrolidine

25.2 g (0.1063 mole) (3RS)-1-bensyl-2-oxo-4-methoxycarbonyl pyrrolidinewas slowly added to a slurry of 6.22 g lithium aluminium hydride in 160ml diethyl ether under an argon-atmosphere. The mixture was stirred overnight and then heated to reflux for one hour. The reaction mixture wascooled to room temperature and 0.2 g of Na₂SO₄×10H₂O was added followedby a slow addition of, in that order, 6 ml water, 18 ml 3.75 MNaOH-solution and 6 ml water. The slurry was dried from excess of waterwith Na₂SO₄/cellite, filtered and evaporated to give (20.3 g) of theproduct.

¹H-NMR (CDCl₃, 300 MHz): δ 1.64-1.77 (m, 1H), 1.93-2.07 (m, 1H),2.27-2.40 (m, 2H), 2.51 (dd, 1H), 2.62 (dd, 1H), 2.82 (m, 1H), 3.52 (dd,1H), 3.59 (s, 2H), 3.67 (dd, 1H), 7.15-7.40 (m, 5H)

(ii) (3RS)-1-benzyl-3-chloromethyl pyrrolidine

To a refluxing solution of 15.3 g (0.08 mole)(3RS)-1-benzyl-3-hydroxymethylpyrrolidine in 220 ml CHCl₃ was slowlyadded a solution of 330 ml thionyl chloride in 60 ml CHCl₃, and thereflux was continued for one hour. The mixture was evaporated and theresidue was dissolved in water.

The water layer was washed with ethyl acetate and then made alkalinewith 0.2 M NaOH-solution. The water layer was extracted three times withethyl acetate and the combined organic layer was dried (Na₂SO₄),filtered and evaporated to give the product in a quantitative yield(16.8 g).

¹H-NMR (CDCl₃, 300 MHz): δ 1.55 (m, 1H), 2.05 (m, 1H), 2.38 (dd, 1H),2.48-2.64 (m, 3H; thereof 2.58 (t, 2H))), 2.73 (dd, 1H), 3.51 (d, 2H),3.60 (s, 2H), 7.2-7.4 (m, 5H)

(iii) (3RS)-1-benzyl-3-cyanomethyl pyrrolidine

16.8 q (0.08 mole) (3RS)-1-benzyl-3-chloromethyl pyrrolidine and 5.88 g(0.12 mole) of sodium cyanide was dissolved in 250 ml dimethylsulfoxide. The mixture was stirred at 60° C. for two days and at roomtemperature for one week. Water was added and the mixture was extractedthree times with ethyl acetate. The combined organic layer was washedwith brine, dried (Na₂SO₄), filtered and evaporated to yield 14.7 g(92%) of the product.

¹H-NMR (CDCl₃, 500 MHz): δ 1.55 (m, 1H), 2.13 (m, 1H), 2.35 (dd, 1H),2.42 (t, 2H), 2.44-2.59 (m, 2H), 2.65 (m, 1H), 2.73 (dd, 1H), 3.61 (s,2H), 7.2-7.4 (m, 5H)

(iv) (3RS)-1-benzyl-3-aminoethyl pyrrolidine

14.7 g (0.0734 mole) (3RS)-1-benzyl-3-cyanomethyl pyrrolidine dissolvedin 220 ml diethyl ether was slowly added to a slurry of 2.94 g oflithium aluminium hydride in 74 ml diethyl ether under an argonatmosphere. The mixture was stirred over night, and 6 ml water, 18 ml3.75 M NaOH-solution and 6 ml water was added to the mixture. The slurrywas dried from excess of water with Na₂SO₄/cellite, filtered by suctionand evaporated to yield 14.84 g (99%) of the product.

¹H-NMR (CDCl₃, 300 MHz): δ 1.41 (m, 1H), 1.51 (q, 2H), 1.90-2.10 (m, 2H;thereof 2.05 (dd, 1H))), 2.18 (m, 1H), 2.43 (m, 1H), 2.55-2.73 (m, 3H),2.80 (apparent t, 1H), 3.58 (apparent d, 2H), 7.15-7.4 (m, 5H)

(v) (3RS)-1-benzyl-3-(N-tert-butyloxycarbonylaminoethyl) pyrrolidine

To a mixture of 14.84 g (0.0726 mole) (3RS)-1-benzyl-3-aminoethylpyrrolidine, 72.6 ml 1M NaOH-solution, 76 ml water and 145 ml THF wasadded 17.44 g (0.08 mole) di-tert-butyl dicarbonate and the mixture wasstirred over night. The solution was concentrated and extracted threetimes with ethyl acetate. The combined organic layer was washed withbrine, dried (Na₂SO₄), filtered and evaporated. The crude product waspurified by flash chromatography using a stepwise gradient ofCH₂Cl₂/MeOH (95/5, 90/10) as eluent to yield 14.69 g (80%) of theproduct.

¹H-NMR (CDCl₃, 300 MHz): δ 1.25-1.65 (m, 12H; thereof 1.40 (s, 9H)),1.90-2.25 (m, 3H), 2.46 (m, 1H), 2.67 (m, 1H), 2.80 (apparent t, 1H),3.09 (m, 2H), 3.59 (s, 2H), 4.60 (bs, NH), 7.15-7.35 (m, 5H)

(vi) (3RS)-3-(N-tert-butyloxycarbonylaminoethyl) pyrrolidine

3.1 q (0.01 mol) (3RS)-1-benzyl-3-(N-tert-butyloxycarbonylaminoethyl)pyrrolidine was hydrogenated at 0.28 MPa over 0.6 g of Pearlman'scatalyst (Pd(OH)₂) in 40 ml ethanol (95%) over night. After filtrationof the catalyst through cellite and evaporation of the solvent ¹H-NMRshowed that the reaction was not completed. Therefore 0.6 g ofPearlman's catalyst was added in 40 ml ethanol (95%) once more and themixture was treated under H₂-atmosphere at 0.28 MPa over night.Filtration through cellite and evaporation of the solvent gave theproduct in a quantitative yield (2.18 g).

MS m/z 214 (M+)

(vii) (3RS)-1-(N-benzyloxycarbonylamidino)-3-aminoethyl pyrrolidine(H—(R,S)Hig(Z))

2.18 g (0.0102 mmole) (3RS)-3-(N-tert-butyloxycarbonylaminoethyl)pyrrolidine and 2.81 g (0.0125 mole)N-benzyloxycarbonyl-S-methylisothiourea was dissolved in 30 ml tolueneand heated to 60-70° C. for eight hours followed by stirring at roomtemperature for one day. 0.3 M KHSO₄-solution was added and the waterlayer was washed with a mixture of the toluene and ethyl acetate andleft for 2 days under which time the Boc group was removed. The acidicwater phase was made alkaline and extracted four times with CH₂Cl₂ Thecombined organic layer was dried (Na₂SO₄), filtered and evaporated toyield 2.0 g (51%) of the title compound.

¹H-NMR (CDCl₃,330 K, 300 MHz): δ 1.45-1.7 (m, 3H), 2.07 (m, 1H), 2.26(m, 1H), 2.74 (t, 2H), 3.00 (apparent t, 1H), 3.33 (apparent q, 1H),3.45-3.80 (m, 2H), 5.12 (s, 2H), 6.72 (bs, 2 NH), 7.15-7.45 (m, 5H)

(4RS)-1,3-diaza-2-tosylimino-4-aminoethyl cyclohexane (H—(R,S)Itp (Ts))(i) (4RS)-1,3-diaza-2-tosylimino-4-carboxy cyclohexane

Prepared using the same method as described in Journal of Org. Chem., p.46, 1971.

(ii) (4RS)-1,3-diaza-2-tosylimino-4-hydroxymethyl cyclohexane

12.9 g (345 mmol) LiAlH₄ was carefully added to a cold slurry (ice bathtemperature) of 9.9 g (33 mmol) of(4RS)-1,3-diaza-2-tosylimino-4-carboxy cyclohexane in 330 mL dry THF.The reaction was stirred at room temperature over night. The reactionmixture was worked up according to Fieser & Fieser, e.g by adding 12.9 gwater, 38.7 g 3.75 M NaOH, 12.9 g water, Na₂SO₄, CH₂Cl₂ and celite tothe mixture, and filtered. Evaporation of the solvent gave 7.0 g (75%)of the desired product.

MS m/z 284 (M⁺+1)

(iii) (4RS)-1,3-diaza-2-tosylimino-4-mesyloxymethyl cyclohexane

2.9 mL MsCl (37.1 mmol) was added carefully to a cold (ice bathtemperature) slurry of 7.0 g (24.7 mmol) of(4RS)-1,3-diaza-2-tosylimino-4-hydroxymethyl cyclohexane in 6.9 mL (49.4mmol) triethylamine and 125 mL CH₂Cl₂. Water was added after 1 h 15 minand the organic phase was separated, dried (Na₂SO₄), filtered andevaporated to give the title compound in quantitative yield.

MS m/z 362 (M⁺+1))

(iv) (4RS)-1,3-diaza-2-tosylimino-4-cyanomethyl cyclohexane

8.9 g (24.7 mmol) of (4RS)-1,3-diaza-2-tosylimino-4-mesyloxymethylcyclohexane and 1.3 g (27.2 mmol) NaCN was dissolved in 75 mL DMSO.After stirring at 40° C. for 60 hours an additional amount of 0.31 g (6mmol) NaCN was added and the solution was stirred at 65° C. for threehours. 150 mL water was added and crystals precipitated out of thesolution. They where filtered off and dried to give 5.4 g (75%) of thedesired product.

MS m/Z 293 (M⁺+1)

(4RS)-1,3-diaza-2-tosylimino-4-aminoethyl cyclohexane (H—(R,S)Itp(Ts))

935 mg LiAlH₄ was added carefully to a cooled (ice bath temperature)slurry of 2.4 g (8.2 mmol) of (4RS)-1,3-diaza-2-tosylimino-4-cyanomethylcyclohexane in 90 mL THF. After stirring for 2 hours 1 g H₂O, 3 g 3.75MNaOH, 1 g H₂O, Na₂SO₄, celite and CH₂Cl₂ was added. The mixture wasfiltered and the solvent removed in vacuo to give 2.2 g (90%) of thedesired product.

¹H NMR (500 MHz, MeOD); δ 1.52-1.71 (m, 3H), 1.88-1.96 (m, 1H), 2.37 (s,3H), 2.64-2.73 (m, 2H), 3.2-3.4 (m, 2H, partially overlapping with thesolventsignal), 3.44-3.53 (m, 1H), 7.28 (d, 2H), 7.71 (d, 2H)

(4S)-1,3-diaza-2-tosylimino-4-aminoethyl cyclohexane (H—(S)Itp(Ts))

Prepared in the same way as described for H—(R,S)Itp(Ts) starting fromoptically pure 2,4-diaminobutyric acid.

¹H NMR (300.13 MHz, CDCl₃); δ 0.97-1.15 (s broad, 1H), 1.48-1.69 (m,3H), 1.84-1.95 (m, 1H), 2.37 (s, 3H), 2.68-2.82 (m, 1H), 2.86-2.98 (m,1H), 3.22-3.44 (m, 2H), 3.45-3.58 (m, 1H), 7.19 (d, 2H), 7.72 (d, 2H)

¹³C NMR (300.13 MHz, CDCl₃); 6 guanidinecarbon 154.05

H-Aze-OEt×HCl

Prepared in the same way as described for H-Pic-OEt×HCl from H-Aze-OH(vide infra).

H-Aze-OMe×HCl

Prepared according to the procedure described by Seebach D. et. al. inLiebigs Ann. Chem., p. 687, 1990.

H-Pab(Z)×KCl

Prepared by adding 1 mole equivalent of 5 M HCl in iso-propanol to asolution of crude H-Pab(Z) in EtOH (about 1 g/10 ml) where uponH-Pab(Z)×HCl immedeately precipitates out of the solution. Afterfiltration the precipitate was washed 2 times with cold EtOH and driedto give the title compound in almost quantitative yield.

H-Pic-OEt×HCl

L-Pipecolinic acid, 4.0 g (0.031 mol), was slurried in 100 ml of abs.ethanol and HCl (g) was carefully bubbled through until a clear solutionwas obtained. It was cooled in an ice bath and 17 ml of thionyl chloridewas added dropwise over 15 min. The ice bath was removed and the mixturewas refluxed for 2.5 h. The solvent was evaporated and the product wasobtained as its hydrochloride salt in a quantitative yield.

¹H-NMR (300 MHz, D₂O): δ 1.33 (t, 3H), 1.8-2.1 (m, 5H), 2.3-2.5 (m, 1H),3.1-3.3 (m, 1H), 3.5-3.7 (m, 1H), 4.14 (dd, 1H), 4.44 (q, 2H).

H—(R,S)betaPic-OMe×HCl

A mixture of 2.0 g (15.5 mmol) nipecotic acid in 8 ml methanol wascooled in an ice-bath and 2.76 g (23.2 mmol) thionyl chloride was added.The mixture was stirred at room temperature for 20 hours. The solventwas evaporated and the residue was dissolved in a small amount ofmethanol, diethylether was added and H—(R,S)betaPic-OMe×HCl precipitatedas white crystals. The crystals 2.57 g (92%) were isolated byfiltration.

Doc-(R)Cgl-OH

Boc-(R)-Pgl-OH, 32.6 g (0.13 mol), was dissolved in 300 ml of methanoland 5 g of Rh/Al₂O₃ was added. The solution was hydrogenated at 5.2 to2.8 MPa for 3 days. After filtration and evaporation of the solvent NMRshowed the presence of about 25% of the methyl ester is of the titlecompound. The crude material was dissolved in 500 ml of THF and 300 mlof water and 20 g of LioH were added. The mixture was stirred overnightand the THF was evaporated. The remaining water phase was acidified withKHSO₄ and extracted three times with ethyl acetate. The combined organiclayer was washed with water, dried (Na₂SO₄) and evaporated to give 28.3g (83%) of the desired product.

¹H-NMR (300 MHz, CDCl₃): δ 0.9-1.7 (m, 20H), 4.0-4.2 (m, 1H), 5.2 (d,1H).

Boc-(R)Cgl-OSu

To an ice-cold solution of 2.01 g (7.81 mmol) of Boc-(R)Cgl-OH and 1.83g (15.6 mmol) of HOSu in 25 ml of CH₃CN was added 1.69 g (8.2 mmol) ofDCC and the reaction was allowed to reach room temperature. Afterstirring for 3 days the precipitated DCU was filtered off and thesolvent evaporated. The residue was dissolved in EtOAc and the organicphase was washed with H₂O, KHSO₄, NaHCO₃, brine and dried(Na₂SO₄).Evaporation of the solvent gave the title compound in quantitativeyield.

Boc-(R)Cha-OSu

Boc-(R)Cha-OH (1 eq.), HOSu (1.1 eq) and DCC or CME-CDI (1.1 eq) weredissolved in acetonitrile (about 2.5 ml/mmol acid) and stirred at roomtemperature over night. The precipitate formed during the reaction wasfiltered off, the solvent evaporated and the product dried in vacuo.(When CME-CDI was used in the reaction the residue, after evaporation ofthe CH₃CN, was dissolved in EtOAc and the organic phase washed withwater and dried). Evaporation of the solvent gave the title compound.

¹H-NMR (500 MHz, CDCl₃, 2 rotamers ca: 1:1 ratio) 0.85-1.1 (m, 2H),1.1-1.48 (m, 4H), 1.5-1.98 (m, 16H; thereof 1.55 (bs, 9H)), 2.82 (bs,4H), 4.72 (bs, 1H, major rotamer), 4.85 (bs, 1H, minor).

Boc-(R)Hoc-OH

Boc-(R)Hop-OH (See above), 3.2 q (11.46 Mmol) was dissolved in methanol(75 ml). Rhodium on activated aluminium oxide (Rh/Al₂O₃), 0,5 g wasadded and the mixture was stirred under a hydrogen atmosphere at 0.41MPa for 18 h. The catalyst was filtered off through hyflo and thesolvent evaporated giving the product in almost quantitative yield.

¹H-NMR (500 MHz, CDCl₃): δ 0.90 (m, 2H), 1.08-1.33 (m, 6H), 1.43 (s,9H), 1.60-1.74 (m, 6H), 1.88 (bs, 1H), 4.27 (bs, 1H).

Boc-(R)Hoc-OSu

Prepared in the same way as described for Boc-(R)Cha-OSu fromBoc-(R)Hoc-OH.

Boc-(R)Pr(3-(S)Ph)—OH

Prepared according to the method described by J. Y. L Chung et al inJournal of Organic Chemistry, No 1, pp. 270-275, 1990.

Boc-(R)Cgl-Aze-OH

(i) Boc-(R)Cgl-Aze-OMe

To a stirred mixture of 3.86 g (15 mmol) Boc-(R)Cgl-OH, 2.27 g (15 mmol)H-Aze-OMe×HCl and 2.75 g (22.5 mmol) DMAP in 40 mL CH₃CN at 5° C. wasadded 3.16 g (16.5 mmol) EDC. The reaction mixture was stirred at roomtemperature for 48 h. The solvent was evaporated and the residue wasdissolved in 150 ml EtOAc and 20 ml H₂O. The separated organic layer waswashed with 2×20 ml 0.5 M KHSO₄, 2×10 ml NaHCO₃(saturated), 1×10 ml H₂O,1×10 ml brine and dried (MgSO₄). Evaporation of the solvent gave 4.91 g(92%) of the title compound which was used without further purificationin the next step.

¹H NMR (500 MHz, CDCl₃, 0.1 g/ml): major rotamer, 0.83-1.35 (m, 5H),1.38 (s, 9H), 1.47-1.84 (m, 6H), 2.18-2.27 (m, 1H), 2.50-2.62 (m, 1H),3.72 (s, 3H), 3.94-4.06 (m, 1H), 4.07-4.15 (m, 1H), 4.39-4.47 (m, 1H),4.68 (dd, J=9.1, J=5.1, 1H), 5.09 (d, J=9.2, 1H).

Resolved peaks from minor rotamer, 2.27-2.35 (m, 1H), 3.77 (s, 3H),3.80-3.87 (m, 1H), 3.88-3.95 (m, 1H), 4.92 (d, J=9.2, 1H), 5.21 (dd,J=9.1, J-5, 1H).

(ii) Boc-(R)Cgl-Aze-OH

The hydrolysis of Boc-(R)Cgl-Aze-OMe was carried out according to theprocedure described for Boc-(R)Cha-Pic-OEt (vide infra). The product wascrystallized from EtOH/acetone/water (1/1/3.95) yield 80%.

¹H-NMR (500 MHz, CDCl₃): δ 0.85-1.3 (m, 5H), 1.40 (s, 9H), 1.5-1.9 (m,6H), 1.95-2.2 (m, 2H), 3.92 (m, 1H), 4.09 (m, 1H), 4.35 (m, 1H), 4.95(m, 1H), 5.16 (bd, 1H).

Boc-(R)Cgl-Pic-OH

(i) Boc-(R)Cgl-Pic-OMe

Pivaloyl chloride (1.0 ml, 8.1 mmol) was added to a solution ofBoc-(R)Cgl-OH (2.086 g, 8.1 mmol) and triethyl amine (1.13 ml, 8.1 mmol)in toluene (25 ml) and DMF (5 ml). A mixture of H-Pic-OMe×HCl (1.46 g,8.1 mmol) and triethyl amine (1.13 ml, 8.1 mmol) in DMF (20 ml) wassubsequently added at ice bath temperature. The reaction mixture wasslowly allowed to warm up to room temperature and after 24 h it wasdiluted with water and extracted with toluene. After washing with 0.3 MKHSO₄, 10% Na₂CO₃ and brine the solvent was removed in vacuo to give2.52 g (81) of colorless oil which was used without furtherpurification.

¹H-NMR (500 MHz, CDCl₃, 2 rotamers, 5:1 ratio) δ 0.8-1.8 (m, 25H), 2.25(d, 1H), 2.75 (t, 1H, minor rotamer), 3.3 (t, 1H), 3.7 (s, 3H), 3.85 (d,1H), 4.3 (t, 1H, minor rotamer), 4.5-4.6 (m, 1H), 5.25 (d, 1H), 5.30 (d,1H).

(ii) Boc-(R)Cgl-Pic-OH

Prepared according to the procedure for hydrolysis of Boc-(R)Cha-Pic-OEt(vide infra) using the product from (i) above. The product wascrystallized from di-isopropyl ether and hexane.

¹H-NMR (500 MHz, CDCl₃, 2 rotamers, 5:1 ratio) δ 0.8-1.8 (m, 25H), 2.3(d, 1H), 2.8 (t, 1H, minor rotamer), 3.3 (t, 1H), 3.9 (d, 1), 4.4 (t_(,)1H, minor), 4.5-4.6 (m, 1H), 5.1 (s, 1H, minor rotamer), 5.3 (d, 1H),5.40 (d, 1H).

Boc-(R)Cgl-Pro-OH

3.59 g (31.24 mmol) of L-proline was mixed with 20 ml water and 1.18 g(29.7 mmol) of sodium hydroxide. 2.8 g (7.8 mmol) of Boc-(R)Cgl-OSu in10 ml DMF was added to the mixture. Because of solubility problem anadditional 30 ml of DMF was added and the reaction mixture was stirredfor three days. The solvent was evaporated and water was added. Thewater phase was washed with ethyl acetate, acidified with 0.3 MKHSO₄-solution and extracted three times with ethyl acetate. The organicphase was washed once with water and once with brine, dried (Na₂SO₄),filtered and evaporated to yield 2.3 g (83%) of the product.

¹H-NMR (300 MHz, CDCl₃): δ 0.89-2.17 (m, 23H), 2.37 (m, 1H), 3.55 (q,1H), 3.90 (bs, 1H), 4.28 (t, 1H), 4.52 (bs, 1H), 5.22 (bs, 1H(NH)).

Boc-(R)Cha-Aze-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH startingfrom Boc-(R)Cha-OH and H-Aze-OEt×HCl (vide infra).

Boc-(R)Cha-Pro-OH

H—(S)Pro-OH (680 mmol) was dissolved in 0.87 M sodium hydroxide (750ml). Boc-(R)Cha-OSu (170 mmol) dissolved in DMF (375 ml) was addeddropwise during 20 min. The reaction mixture was stirred at roomtemperature for 20 h. The mixture was acidified (2M KHSO₄) and extractedthree times with ethyl acetate. The organic layers were combined andwashed three times with water and once with brine. After drying oversodium sulphate and evaporation of the solvent, the syrupy oil wasdissolved in diethyl ether, the solvent evaporated and finally theproduct dried in vacuo to yield Boc-(R)Cha-Pro-OH as a white powder inalmost quantitative yield.

¹H-NMR (500 MHz, CDCl₃, mixture of two rotamers 9:1) δ 0.8-1.05 (m, 2H),1.05-1-55 (m, 15H; thereof 1.5 (bs, 9H)), 1.55-1.8 (m, 5H), 1.8-2.15 (m,3H), 2.47 (m, 1H), 3.48 (m, 1H), 3.89 (m, 1H), 4.55 (m, 2H), 5.06 (m,1H);

Resolved signals from the minor rotamer appears at d 2.27 (m), 3.58 (m),4.33 (m), 5.0 (m).

Boc-(Me)(R)Cha-Pro-OSu

(i) Boc-(Me)(R)Cha-Pro-OH

Prepared in the same way as described above for Boc-(R)Cha-Pro-OHstarting from Boc-(Me)(R)Cha-OSu and H-Pro-OH.

(ii) Boc-(Me)(R)Cha-Pro-OSu

Prepared in the same way as described for Boc-(R)Cha-OSu starting fromBoc-(Me)(R)Cha-Pro-OH.

Boc-(R)Cha-Pic-OH

(ia) Boc-(R)Cha-Pic-OEt

Boc-(R)Cha-OH, 6.3 g (0.023 mol), was dissolved in 150 ml of CH₂Cl₂. Thesolution was cooled in an ice bath and 6.3 g (0.047 mol) ofN-hydroxybenzotriazole and 11.2 g (0.0265 mol) of CME-CDI were added.The ice bath was removed after 15 min and the reaction mixture wasstirred for 4 h at room temperature. The solvent was evaporated and theresidue dissolved in 150 ml of DMF and cooled in an ice bath.H-Pic-OEtxHCl, 4.1 g (0.021 mol) was added and the pH adjusted toapproximately 9 by addition of N-methylmorpholine. The ice bath wasremoved after 15 min and the reaction mixture was stirred for 3 days.The solvent was evaporated and the residue was dissolved in ethylacetate and washed with dilute KHSO₄ (aq), NaHCO₃ (aq) and water. Theorganic layer was dried (Na₂SO₄) and evaporated to give 7.7 g (89%) ofBoc-(R)Cha-Pic-OEt which was used without further purification.

¹H-NMR (500 MHz, CDCl₃, 2 rotamers, 3:1 ratio) d 0.7 -1.0 (m, 2H),1.1-1.9 (m, 29H; thereof 1.28 (t, 3H)), 1.45 (bs, 9H), 2.01 (bd, 1H,major rotamer), 2.31 (bd, 1H), 2.88 (bt, 1H, minor), 3.30 (bt, 1H,major), 3.80 (bd, 1H, major), 4.15-4.3 (m, 2H), 4.5-4.7 (m, 2H, minor),4.77 (bq, 1H, major), 4.90 (bd, 1H, minor), 5.28 (bd, 1H, major), 5.33(bd,1H, major).

(ib) Boc-(R)Cha-Pic-OMe

400 μl (3.23 mmol) of pivaloyl chloride was added to a stirred mixtureof 875 mg (3.22 mmol) Boc-(R)Cha-OH and 450 μl (3.23 mmol) triethylamine in 10 ml toluene and 2 ml DMF. A mixture of 596 mg (3.32 mmol)methyl(S)-pipecolate hydrochloride and 463 μl (3.32 mmol) triethyl aminein 5 ml DMF was added to the resulting slurry after 45 minutes. After 2h 100 μl (0.72 mmol) triethyl amine was added and stirring was continuedfor another 18 h. Water and toluene was added to the reaction mixtureand the organic phase was washed with 0.3 M KHSO₄, 10% Na₂CO₃ and brine.Drying (MgSO₄) and removal of the solvent in vacuo gave 1.16 g of thetitle compound.

(ii) Boc-(R)Cha-Pic-OH

Boc-(R)Cha-Pic-OEt, 5.6 g (0.014 mol), was mixed with 100 ml of THF, 100ml of water and 7 g of LiOH. The mixture was stirred at room temperatureovernight. The THF was evaporated and the aqueous solution was acidifiedwith KHSO₄ (aq) and extracted three times with ethyl acetate. Thecombined organic phase was washed with water, dried (Na₂SO₄) andevaporated to give 4.9 g (94%) of Boc-(R)Cha-Pic-OH which was usedwithout further purification. The compound can be crystallized fromdiisopropyl ether/hexane.

The methyl ester formed in procedure (ib) above can be hydrolysed usingthe same procedure as described for the ethyl ester in (ii).

¹H-NMR (500 MHz, CDCl₃, 2 rotamers, 3.5:1 ratio) δ 0.8-1.1 (m, 2H),1.1-2.1 (m, 27H; thereof 1.43 (s, 9H, major rotamer), 1.46 (s, 9H,minor)), 2.33 (bd, 1H), 2.80 (bt, 1H, minor), 3.33 (bt, 1H, major), 3.85(bd, 1H, major), 4.57 (bd, 1H, minor), 4.68 (m, 1H, minor), 4.77 (bq,1H, major), 5.03 (bs, 1H, minor), 5.33 (bd, 1H, major), 5.56 (m, 1H,major).

Boc-(R)Cha-(R,S)betapic-OH

(i) Boc-(R)Cha-(R,S) betaPic-OMe

Pivaloyl chloride 0.9 ml (7.3 mmol) was added to a solution of 2.0 g(7.3 mmol) Boc-(R)Cha-OH and 0.81 ml (7.3 mmol) 4-N-methyl morpholin in20 ml acetonitrile. After stirring for 1 h and 30 minutes 1.3 g (7.3mmol) H—(R,S)betaPic-OMe×HCl and 1.62 ml (14.6 mmol) 4-N-methylmorpholine was added and the reaction mixture was stirred for 24 h. Thesolvent was evaporated and the residue was dissolved in toluene and somediethyleter. After washing with 0.3 M KHSO₄ and KHCO₃-solution, anddrying with Na₂SO₄ the solvent was removed in vacuo. Flashchromatography using heptane/ethyl acetate (7/3) as eluent gave 2.4 g(83%) of the desired product.

(ii) Boc-(R)Cha-(R,S)betaPic-OH

At room temperature 2.35 g (5.9 mmol) of Boc-(R)Cha-(R,S)betaPic-OMe wasdissolved in 35 ml THF and 2.1 g of LiOH in 35 ml water was added. Afterstirring for 5 h the THF was removed in vacuo. The aqueous phase wasacidified with 2M KHSO₄ and extracted with ethyl acetate, dried overNa₂SO₄ and evaporated to give 2.0 g (89%) of the product.

Boc-(R)Cha-Val-OH

(i) Boc-(R)Cha-Val-OMe

3.1 ml (25 mmol) pivaloyl chloride was added at ambient temperature to astirred mixture of 6.75 g (25 mmol) Boc-(R)Cha-OH and 3.5 ml (25 mmol)triethyl amine in 50 ml DMF. After 3 hours 4.16 g (25 mmol) valinemethyl ester hydrochloride in 50 ml DMF and 3.5 ml triethyl amine wasadded. After stirring over night, a few crystals of DMAP were added andthe reaction mixture was heated to 50° C. for 5 minutes. The solvent wasremoved in vacuo and ether and toluene was added to the residue. Washingwith 0.3 N KHSO₄ and 10% Na₂CO₃ followed by drying (MgSO₄) and removalof the solvent in vacuo gave a residue which was subjected to flashchromatography using toluene/ethyl acetate as eluent. The yield of thetitle compound was 6.99 g (73%).

(ii) Boc-(R)Cha-Val-OH

A mixture of 8.73 g (23 mmol) Boc-(R)Cha-Val-OMe and 5.6 g (230 mmol)lithium hydroxide in 75 ml THF and 75 ml of water was stirred for 4hours. The THF was removed in vacuo and the remaining solution wasdiluted with water and extracted with ether. Acidification with 2 MKHSO₄ and extraction with ethyl acetate followed by drying (MgSO₄) andremoval of the solvent in vacuo gave 8.15 g (96%) of the title compound.

Boc-(R)Hoc-Aze-OH

(i) Boc-(R)Hoc-Aze-OEt

At room temperature 1.0 g (3.5 mmol) Boc-(R)Hoc-OH and 0.95 g (7.0 mmol)HOBt was dissolved in 15 ml CH₂Cl₂. The solution was cooled in an icebath and 0.77 g (4.0 mmol) of EDC was added. The ice bath was removedand the reaction mixture was stirred for 3 h at room temperature. Thesolvent was evaporated and the residue dissolved in 20 ml DMF. 0.58 g(3.5 mmol) H—(R)Aze-OH was added and the pH adjusted to approximately 9by addition of N-methyl morpholin. The reaction mixture was stirred forone day. The reaction mixture was partitioned between water and toluene.The organic phase was separated and washed with 0.3 M KHSO₄, dilutedKHCO₃, brine, dried with NaSO₄ and evaporated. Flash chromatography (1%EtOH in CH₂Cl₂ and heptane: EtOAc) gave 0.35 g (25%) of the desiredproduct.

(ii) Boc-(R)Hoc-Aze-OH

At room temperature 0.65 g (1.6 mmol) Boc-(R)Hoc-Aze-OEt was dissolvedin 10 ml THF and 0.59 g of LiOH in 10 ml water was added. After stirringfor 24 hours 2 M KHSO₄ was added and the THF was removed in vacuo. Theaqueous phase was then made acidic with more 2M KHSO₄ and extracted withethyl acetate, dried over Na₂SO₄ and evaporated to give 0.5 g (85%) ofthe title compound.

Boc-(R)Roc-Pro-OH

Prepared in the same way as described for Boc-(R)Cha-Pro-OH fromBoc-(R)Hoc-OSu.

¹H-NMR (500 MHz, CDCl₃): δ 0.80-0.94 (m, 2H), 1.05-1.36 (m, 7H),1.36-1.48 (bs, 9H), 1.48-1.78 (m, 7H), 1.98-2.14 (m, 2H), 2.34 (m, 1H),3.48 (m, 1H), 3.85 (m, 1H), 4.43 Cm, 1H), 4.52 (bd, 1H), 5.26 (bd, 1H),signals of a minor rotamer appears at: δ 1.92, 2.25, 3.58, 4.20 and4.93.

Boc-(R)Hoc-Pic-OH

(i) Boc-(R)Hoc-Pic-OMe

Prepared the same way as described for Boc-(R)Cha-Pic-OEt (vide supra)from Boc-(R)Hoc-OH and H-Pic-OMe×HCl.

(ii) Boc-(R)Hoc-Pic-OH

Prepared in the same way as described for Boc-(R)Cha-Pic-OH (vide supra)from Boc-(R)Hoc-Pic-OMe.

¹H-NMR (500 MHz, CDCl₃): δ 0.82-0.97 (m, 2H), 1.10-1.36 (m, 7H),1.36-1.50 (bs, 9H), 1.50-1.82 (m, 11H), 2.35 (bd, 1H) 3.28 (bt. 1H),3.85 (bd, 1H) 4,63 (m, 1H), 5.33 (bs, 1H), 5.44 (bd, 1H), signals of aminor rotamer appear at: δ 1.88, 2.80, 4.25, 4.55 and 4.97.

Boc-(R)Pro-Phe-OH

(i) Boc-(R)Pro-Phe-OMe

To a solution of 2.0 q (9.29 mmol) Boc-(R)Pro-OH and 0.94 g (9.29 mmol)triethyl amine in 70 ml toluene/DMF (5/2) was added 1.12 g (9.29 mmol)pivaloylchloride and the reaction was stirred for 30 minutes at roomtemperature. The reaction was cooled to 0° C. and a mixture of 2.0 g(9.29 mmol) H-Phe-OMe and 0.94 g triethyl amine in 40 ml DMF was addedand the reaction was stirred over night at room temperature. Thereaction mixture was diluted with toluene and the organic phase waswashed with 3×50 ml 0.3 M KHSO₄, 1×50 ml water and dried (Na₂SO₄).Evaporation of the solvent gave the title compound in quantitative yieldwhich was used in the next step without further purification.

(ii) Boc-(R)Pro-Phe-OH

A mixture of 4.0 g (10.6 mmol) Boc-(R)Pro-Phe-OMe and 8.93 g (21.3 mmol)LiOH×H₂O in 140 ml water/THF (1/1) was stirred vigorously over night atroom temperature. The THF was evaporated and the water phase was madeacidic with 1 M KHSO₄ and extracted with 3×75 ml EtOAc. The combinedorganic phase was washed with water and dried (Na₂SO₄). Filtration andevaporation of the solvent gave a residue which was purified bycrystallization from diisopropyl ether to give 2.329 g (60%) of thetitle compound as a white crystalline solid.

Boc-(R)Pro(3-(S)Ph)-Pro-OH

(i) Boc-(R)Pro(3-(S)Ph)-Pro-OBn

To a mixture of 1.61 g Boc-(R)Pro(3-(S)Ph)—OH, 1.65 g H-Pro-OBn×HCl and0.75 g HOBt in 11 mL DMF was added 0.84 mL NMM and 2.92 g CME-CDI atroom temperature and the reaction mixture was stirred for three days.The solvent was evaporated and the residue was dissolved in 300 mLEtOAc. The organic phase was washed with 2×100 mL H₂O, 2×100 mL 1 MKHSO₄, 3×100 mL 1 M NaOH, 3×100 ml H₂O and dried (MgSO₄). Evaporation ofthe solvent gave 2.53 g of an oil which was purified by flashchromatography using CH₂Cl₂/MeOH (97/3) as eluent to give 2.11 g (88%)of the title compound.

(ii) Boc-(R)Pro(3-(S)Ph)-Pro-OH

0.94 q of Boc-(R)Pro(3-(S)Ph)-Pro-OBn was dissolved in 70 ml EtOH andhydrogenated over 0.42 g 5% Pd/C for 3.5 hours. Filtration of thecatalyst and evaporation of the solvent gave the title compound as whitecrystals in a quantitative yield.

Boc-(R)Tic-Pro-OH

Prepared according to the procedure described by P. D. Gesellchen and R.T. Shuman in EP-0,479,489-A2.

BnOOC—CH₂—NH—CO—CH₂—Br

To a solution of p-TsOH×H-Gly-OBn (5 mmol) and triethyl amine (5 mmol)in 10 ml of CH₂Cl₂ was added 2-bromoacetic acid (5 mmol) dissolved in 10ml of CH₂Cl₂ and dicyclohexyl carbodiimide (5 mmol). The mixture wasstirred at room temperature over night and filtered. The organic phasewas washed twice with 0.2 M KHSO₄, 0.2 M NaOH, brine and dried.Evaporation and flash chromatography (CH₂Cl₂/MeOH, 95/5) gave aquantitative yield of the desired compound.

¹H-NMR (300 MHz, CDCl₃): δ 3.89 (s, 2H), 4.05-4.11 (d, 2H), 5.19 (s,2H), 7.06 (bs, 1H), 7.3-7.4 (m, 5H).

Boc-(R)Cgl-Ile-OH

Prepared in the same way as described for Boc-(R)Cgl-Pro-OH usingH-Ile-OH, instead of H-Pro-OH, in 91% yield.

Boc-(R)Phe-Phe-OH

(i) Boc-(R)Phe-Phe-OMe

Boc-(R)Phe-OH (18.8 mmol; purchased from Bachem Feinchemicalien AG),Phe-OMe (20.7 mmol) and 4-dimethylaminopyridine (37.7 mmol) weredissolved in 30 mL of acetonitrile. The solution was cooled to ice-watertemperature and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (24.5 mmol) was added. The cooling bath was removed andthe reaction mixture was stirred over night. The solvent was thenremoved under reduced pressure and the residue was dissolved in 50 mL ofethylacetate. Extraction of the organic phase with 50 mL aliquats of 0.5M potassiumhydrogensulfate, 1 M sodiumbicarbonate and finally waterfollowed by evaporation of the solvent yielded 7.5 g ofBoc-(R)Phe-Phe-OMe (94%) which was used in the next step without furtherpurification.

(ii) Boc-(R)Phe-Phe-OH

Boc-(R)Phe-Phe-OMe (16.4 mmol) was dissolved in 40 mL of tetrahydrofuranand lithiumhydroxide (32.8 mmol) dissolved in 20 mL of water was addedrapidly. The reaction mixture was stirred for 3.5 h after which thesolvent was removed under reduced pressure. The residue was dissolved in50 mL of ethylacetate and extracted with 50 mL of 0.5 M potassiumsulfatefollowed by 50 mL of water. The solvent was removed under reducedpressure yielding 8.0 g of Boc-(R)Phe-Phe-OH (quant) as an amorphoussolid. ¹H NMR (200 MHz, d-CHCl₃); δ 7.4-6.7 (m, 10H), 5.7-4.2 (m, 6H),1.34 (s, 9H).

HO—CH₂—COOBn

Prepared according to the procedure described by Lattes A. et al inBull. Soc. Chim. France., Noll, pp 4018-23, 1971.

Benzyl-2-(ortho-nitrobenzenesulfonyloxy)acetate(2-NO₂) Ph-SO₂—OCH₂—COOBn

1.66 g (10 mmol) benzylglykolate was dissolved in 25 ml CH₂Cl₂ and 25 mldiethylether. The mixture was cooled to 0° C. and 2.8 ml (20 mmol)triethylamin was added. While keeping the temperature at 0° C. 2.44 g(11 mmol) orto-nitrobenzenesulfonylchloride was added in small portionsduring 15 minutes. The slurry was stirred at OC for 50 minutes and then20 ml water and 30 ml CH₂Cl₂ were added. The phases were separated andthe organic phase was washed with 20 ml 1 M HCl and 20 ml H2O, dried(Na₂SO₄), filtered and evaporated in vacuo to give 3.34 g of a residuethat was subjected to flash chromatography, using heptan:EtOAc 2:1 aseluent to give 1.18 g (34%) of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ 4.92 (s, 2H), 5.17 (s, 2H), 7.83 (m, 5H),7.76 (m, 3H), 8.16 (dd, 1H).

Benzyl-2-(para-nitrobenzenesulfonyloxy)acetate(4-NO₂) Ph—SO₂—OCH₂—COOBn

Prepared according to the same procedure as described forBenzyl-2-(ortho-nitrobenzenesulfonyloxy)acetate above. The finalcompound was obtained in a crystalline form after evaporation of thesolvent and pure enough to use without further purification (64% yield).

¹H-NMR (300 MHz, CDCl₃): δ 4.79 (s, 2H), 5.13 (s, 2H), 7.2-7.4 (a, 5H),8.10 (d, 2H), 8.30 (d, 2H).

TfO—CH₂COOMe

10.09 ml (60 mmol) trifluorometansulfonic anhydrid dissolved in CH₂Cl₂was added dropwise to a mixture of 4.05 ml (50 mmol) methylglycolate and4.04 ml (50 mmol) pyridin in CH₂Cl₂ (totally 62.5 ml) at 0° C. during 25minutes, and thereafter stirred at 0° C. for 1H. After washing with 0.3M KHSO₄ and saturated NA₂CO₃, drying (Na₂SO₄) and filtration,evaporation of the solvent in vacuo gave 9.94 g (90%) of the titlecompound.

TfO-CR₂COOEt

Prepared in the same way as described for TfO-CH₂COOMe starting withethylglycolate.

TfO—CH₂COO^(n)Bu

Prepared in the same way as described for TfO-CH₂COOMe starting withbutylglycolate.

TfO—CH₂COOBn

Prepared in the same way as described for TfO-CH₂COOMe starting withHO—CH₂COOBn

TfO-CH₂COO^(n)Hax

(i) HO—CH₂COO^(n)Hex

To 215 mg (2.82 mmol) glycolic acid in 12.8 ml CH₃CN was added 719 mg(3.39 mmol) 1-hexyl iodide and 429 mg (2.82 mmol) DBU. After stirringover night and reflux for 4 h, the solvent was evaporated, ethylacetatand 1 M KHSO₄ was added and the phases were separated. The organic layerwas washed with brine, dried (MgSO₄), filtered and evaporated in vacuoto give 333 mg (74%) of the product.

(ii) TfO-CH₂COO^(n)Hex

Prepared in the same way as described for TfO-CH₂COOMe starting withHO—CH₂COO^(n)Hex.

H-Mig(z) (3-aminomethyl-1-(N-benzyloxycarbonylamidino) azetidine (i)3-aminomethyl-1-benzhydryl azetidine was Prepared According to theLiterature, see A. G. Anderson, Jr., and R. Lok, J.Org.Chem., 37, 3953,1972. (ii) 3-(N-tert-butyloxycarbonylaminomethyl)-1-benzhydryl azetidine

To 3.50 g (13.9 mmol) of 3-aminomethyl-1-benzhydryl azetidine dissolvedin 45 mL THF was added a solution of 0.56 g (13.9 mmol) NaOH in 45 mLH₂O. The reaction mixture was cooled to 0° C. and 3.03 g (13.9 mmol) ofdi-tert-butyl dicarbonate was added. The cooling bath was removed aftera few minutes and the mixture was stirred at roomtemperature over night.The THF was evaporated and the residue was extracted with 3×45 mL ofdiethyl ether. The combined organic layer was washed with brine, driedwith Na₂SO₄ and filtered. Evaporation of the solvent gave 4.6 g (94%) ofthe title compound.

(iii) 3-(N-tert-butyloxycarbonylaminomethyl) azetidine

3.4 g (9.6 mmol) of 3-(N-tert-butyloxycarbonylaminomethyl)-1-benzhydrylazetidine was dissolved in 170 mL MeOH and hydrogenated over 0.30 gPd(OH)₂ at 5 MPa over night. The catalyst was filtered off and thesolvent evaporated. The crude product was purified by flashchromatography using MeOH/CH₂Cl₂, 1/9, followed by MeOH (saturated withNH₃ (g))/CH₂Cl₂₁ 1/9, as eluent to yield 1.2 g (67%) of the titlecompound.

(iv)3-(N-tert-butyloxycarbonylaminomethyl)-1-(N-benzyloxycarbonylamidino)azetidine (Boc-Mig(Z))

0.9 g (4.8 mmol) of 3-(N-tert-butyloxycarbonylaminomethyl) azetidine and1.3 g (6.3 mmol) of N-benzyloxycarbonyl-O-methylisourea was mixed in 6.5mL toluene and heated to 70° C. for 72 h and then left atroomtemperature for another 72 h. Evaporation followed by flashchromatography using EtOAc followed by MeOH (saturated withNH₃(g))/CH₂Cl₂₁ 1/9, as eluent gave 0.67 g (38%) of the title compoundas a white powder.

(v) 3-aminomethyl-1-(N-benzyloxycarbonylamidino) azetidine (H-Mig(Z))

0.67 g (1.85 mmol) of Bcc-Mig(Z) was dissolved in 10 mL of EtOAcsaturated with HCl(g) and stirred for 10 min. at roomtemperature. 10 mLof a saturated solution of KOH(aq) was added dropwise. The layers wereseparated and the aqueous phase was extracted with 3×8 mL EtOAc. Theorganic layers were combined, washed with brine, dried with Na₂SO₄ andevaporated to yield 0.43 g (89%) of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ 2.55-2.65 (m, 1H), 2.84 (d, 2H), 3.66 (dd,2H) 4.03 (dd, 2H) 5.07 (s, 2H), 7.2-7.4 (m, 5H).

MS m/z 263 (M⁺+1)

3-amino thyl-1-(N-benzyloxycarbonylamidino) azetidine (H-Dig(Z)) (i)3-carboxylic acid-1-benzhydryl azetidine was Prepared According to theLiterature, see A. G. Anderson, Jr., and R. Lok, J.Org.Chem., 37, 3953,1972. (ii) 3-hydroxymethyl-1-benzhydryl azetidine

A solution of 8.7 g (32.5 mmol) 3-carboxylic acid-1-benzhydryl azetidinein 80 mL of dry THF was added slowly to a suspention of 4.9 g (130.2mmol) of LiAlH₄ in 30 mL THF at roomtemperature. The reaction mixturewas refluxed for 3.5 h. Excess hydride reagent was hydrolyzed by carefuladdition, with cooling, of NH₄Cl(aq), the gelatinous mixture wasfiltered and the filter cake was washed repeatedly with THF. Evaporationof the solvent gave 7.1 g (86%) of the title compound as pale yellowcrystals.

(iii) 3-methanesulfonatomethyl-1-benzhydryl azetidine

To a solution of 6.62 g (26.1 mmol) 3-hydroxymethyl-1-benzhydrylazetidine in 50 mL of dry pyridine was added 4.50 g (39.2 mmol) ofmethanesulfonyl chloride at 0° C. The reaction mixture was stirred for 1h. and then allowed to stand in a refrigerator over night. The reactionmixture was poured into a mixture of ice and H₂O. The precipitate wascollected, washed with H₂O and dried in vacuo to yield 7.75 g (89.5%) ofthe title compound.

(iv) 3-cyanomethyl-1-benzhydryl azetidine

To a solution of 7.75 g (23.4 mmol)3-methanesulfonatomethyl-1-benzhydryl azetidine in 50 mL DMF was added asolution of 3.44 g (70.0 mmol) NaCN in 10 mL H₂O. The mixture was heatedat 65° C. for 20 h, cooled, and poured into a mixture of ice and H₂O.The precipitate was collected, washed with H 0 and dried in vacuo toyield 5.7 g (93%) of the title compound.

(v) 3-aminoethyl-1-benzhydryl azetidine

5.7 g (21.7 mmol) of 3-cyanomethyl-1-benzhydryl azetidine was addedslowly to a suspention of 2.9 g (76.0 mmol) of LiAlH₄ in 80 mL of dryTHF at roomtemperature. The reaction mixture was refluxed for 4 h.Excess hydride reagent was hydrolyzed by careful addition, with cooling,of NH₄Cl(aq), the gelatinous mixture was filtered and the filter cakewas washed repeatedly with THF. The solvent was evaporated, the residuewas dissolved in diethyl ether, washed with brine and dried with Na₂SO₄.Evaporation of the solvent gave 5.0 g (87%) of the title compound.

(vi) 3-(N-tert-butyloxycarbonylaminoethyl)-1-benzhydryl azetidine

The title compound was prepared from 3-aminoethyl-1-benzhydryl azetidineaccording the procedure for3-(N-tert-butyloxycarbonylaminomethyl)-1-benzhydryl azetidine, in ayield of 6.5 g (95%).

(vii) 3-(N-tert-butyloxycarbonylaminoethyl)azetidine

The title compound was prepared from3-(N-tert-butyloxycarbonylaminoethyl)-1-benzhydryl azetidine accordingthe procedure for 3-(N-tert-butyloxycarbonylaminomethyl) azetidine, in ayield of 1.2 g (70%).

(viii) 3-(N-tert-butyloxycarbonylaminoethyl)-1-(N-benzyloxycarbonylamidino) azetidine (Boc-Dig(Z))

The title compound was prepared from3-(N-tert-butyloxycarbonylaminoethyl) azetidine according the procedurefor 3-(N-tert-butyloxycarbonylaminomethyl)-1-(N-benzyloxycarbonylamidino) azetidine, in a yield of 0.090 g (34%).

(ix) 3-aminoethyl-1-(N-benzyloxycarbonylamidino) azetidine (H-Dig(Z))

0.589 g (1.56 mmol) of Boc-Dig(Z) was dissolved in 10 mL of EtOAcsaturated with HCl(g) and stirred for 10 min. at roomtemperature. 10 mLof a saturated solution of KOH(aq) was added dropwise. The layers wereseparated and the aqueous phase was extracted with 3×8 mL EtOAc. Theorganic layers were combined, washed with brine, dried with Na₂SO₄ andevaporated to yield 0.415 g (96%) of the title compound.

¹H-NMR (500 MHz, CDCl₃): δ 1.60 (dt, 2H), 2.52-2.54 (m, 3H), 3.53 (bs,2H), 4.0 (bt, 2H), 5.00 (s, 2H), 7.17-7.31 (m, 5H).

WORKING EXAMPLES Example 1

HOOC—CH₂—(R)Cgl-Aze-Pab

(i) Boc-(R)Cgl-Aze-Pab(Z)

To a stirred mixture of 3.40 g (10 mmol) Boc-(R)Cgl-Aze-OH (SeePreparation of starting materials) and 5.13 g DMAP (42 mmol) in 120 mlCH₃CN was added 3.18 g H-Pab(Z)×HCl (See Preparation of startingmaterials). After stirring for 2 hours at room temperature the mixturewas cooled to −8° C. and 2.01 g (10.5 mmol) EDC was added. The reactionwas allowed to reach room temperature and the stirring was continued foran additional 47 hours. The solvent was evaporated and the residue wasdissolved in 200 ml EtOAc. The organic phase was washed with 1×50 mlwater, 1×50+2×25 ml 0.5 M KHSO₄, 2×25 ml NaHCO₃(saturated), 1×50 mlwater and dried. Evaporation of the solvent gave 5.21 g (86%) of thetitle compound.

¹H-NMR (500 MHz, CDCl₃): δ 0.8-1.9 (m, 20H; thereof 1.30 (s, 9H)),2.35-2.6 (m, 2H), 3.74 (bt, 1H), 4.10 (m, 1H), 4.25-4.4 (m, 2H),4.45-4.6 (m, 1H, rotamers), 4.75-5.0 (m, 1H, rotamers), 5.08 (bd, 2H),5.15 (s, 2H), 7.15-7.35 (m, 5H), 7.41 (d, 2H), 7.77 (d, 2H), 8.21 (m,1H).

(ii) H—(R)Cgl-Aze-Pab(Z)

To a cold (ice bath temperature) solution of 18.8 g HCl_((g)) in 195 mlEtOAc was added 4.69 g (7.743 mmol) of Boc-(R)Cgl-Aze-Pab(Z) togetherwith 40 ml EtOAc. The reaction mixture was allowed to reach roomtemperature and stirred for 30 min. 140 ml Et₂O was added to the clearsolution where upon a precipitate was formed. The reaction was left atroom temperature for an additional 1 h and 40 minutes. The precipitatewas filtered off, washed quickly with 150 ml Et₂O and dried in vaccuo.The precipitate was dissolved in 50 ml of water and made alkaline with15 ml 2 M NaOH. The alkaline waterphase was extracted with 1×100+1×50 mlCH₂Cl₂. The combined organic phase was washed with 1×20 ml water, 1×20ml Brine and dried (MgSO₄). Evaporation of the solvent gave 3.44 g (88%)of the title compound.

¹H-NMR (500 MHz, CDCl₃): δ 0.8-2.0 (m, 11H), 2.51 (m, 1H), 2.67 (m, 1H),3.07 (d, 1H), 4.11 (m, 1H), 4.18 (m, 1H), 4.43 (dd, 1H), 4.53 (dd, 1H),4.91 (m, 1H), 5.22 (s, 2H), 7.2-7.4 (m, 7H), 7.45 (d, 2H), 8.51 (d, 2H).

(iii) BnOOC—CH₂—(R)Cgl-Aze-Pab(Z)

1.13 g (2.2 mmol) H—(R)Cgl-Aze-Pab(Z), 0.9 g (2.6 mmol)benzyl-2-(orto-nitrobenzenesulfonyloxy)acetate((2-NO₂)Ph—SO₂—OCH₂—COOBn)(See Preparation of starting materials), 0.99 g (5.6 mmol) K₂CO₃ and 113ml CH₃CN were mixed and heated in a 60° C. oilbath for 3 h. The solventwas evaporated in vacuo. EtOAc was added and the mixture was washed withwater, the organic phase was extracted with 1 M KHSO₄ and thiswaterphase was washed with EtOAc. The acidic waterphase was madealcaline with 1 N NaOH to pH>8 and extracted with EtOAc. The organicphase was washed with water, dried (Na₂SO₄), filtered and evaporated invacuo to give 1.17 g of a residue that was twice subjected to flashchromatography using first CH₂Cl₂/MeOH(NH₃-saturated) 95/5 and thendiethylether/MeOH(NH₃-saturated) 9/1 as eluents to give 0.525 g (36%) ofthe title compound.

The alkylation was also carried out usingBenzyl-2-(para-nitrobenzenesulfonyloxy)acetate((4-NO₂)Ph-SO₂—OCH₂—COOBn)(See Preparation of starting materials) using the same procedure asabove to give the title compound in 52% yield.

¹H-NMR (300 MHz, CDCl₃): δ 0.85-2.15 (m, 1H), 2.48 (m, 1H), 2.63 (m,1H), 2.88 (d, 1H), 3.24 (d, 1H), 3.27 (d, 1H), 3.95 (m, 1H), 4.05 (m,1H), 4.44 (m, 1H), 4.55 (m, 1H), 4.91 (m, 1H), 5.07 (s, 2H), 5.22 (s,2H), 7.2-7.4 (m, 10H), 7.45 (d, 2H), 7.79 (d, 2H), 8.42 (m, 1H).

(iva) HOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

BnOOC—CH₂—(R)Cgl-Aze-Pab(z), 20 mg (0.031 mmol), was dissolved in 5 mlof methanol. A few drops of chloroform and 5% Pd/C were added and themixture was hydrogenated at atmospheric pressure for 1 h. Afterfiltration and evaporation the product was lyophilized from water togive 11 mg (72%) of the title compound.

¹H-NMR (500 MHz, D₂O): δ 1.0-2.0 (m, 11H), 2.10 (m, 1H), 2.44 (m, 1H),2.82 (m, 1H), 3.90 (s, 2H), 4.09 (d, 1H), 4.4-4.55 (m, 2H), 4.66 (s,2H), 5.08 (m, 1H), 7.65 (d, 2H), 7.89 (d, 2H).

¹³C-NMR (75.5 MHz, D₂O): amidine and carbonyl carbons: δ 167.3, 167.9,169.9 and 172.4.

(ivb) HOOC—CH₂—(R)Cgl-Aze-Pab

BnOOC—CH₂—(R)Cgl-Aze-Pab(Z) was dissolved in EtOH (99%) and hydrogenatedover 5% Pd/C at atmospheric pressure for 5 hours. Filtration of thecatalyst through cellite and evaporation of the solvent gave the titlecompound in 97% yield.

¹H-NMR (500 MHz, CD₃OD, mixture of two rotamers): major rotamer: δ1.00-1.12 (m, 1H), 1.13-1.34 (m, 4H), 1.55-1.70 (m, 3H), 1.73-1.85 (m,2H), 1.94-2.02 (bd, 1H), 2.32-2.42 (m, 1H), 2.54-2.64 (m, 1H), 2.95-3.10(AB-system plus d, 3H), 4.18-4.25 (bq, 1H), 4.28-4.32 (b, 1H), 4.43-4.60(AB-system, 2H), 4.80-4.85 (dd, 1H), 7.48-7.54 (d,2H), 7.66-7.71 (d,2H).

Resolved signals from the minor rotamer appears at δ 0.95 (m), 1.43 (m),2.24 (m), 2.84 (d), 3.96 (m), 4.03 (m), 7.57 (bd), 7.78 (bd).

¹³C-NMR (125 MHz, CD₃OD): amidine and carbonyl carbons: δ 168.0, 173.0,176.3 and 179.0

Example 2

HOOC—CH₂—CH₂—(R)Cgl-Aze-Pab×2 HCl

(i) H—(R)Cgl-Aze-Pab(Z)

Prepared in the same way as decribed in Example 1 (ii) by treating theformed hydrochloride salt with base to afford the free base.

(ii) BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pab(Z)

H—(R)Cgl-Aze-Pab(Z), 0.19 g (0.38 mmol), and 70 mg (0.43 mmol) of benzylacrylate were dissolved in 2 ml of isopropanol. The mixture was leftstanding for 6 days. Flash chromatography using CH₂Cl₂/THF=8/2 as eluentafforded 0.12 g (48%) of the title compound.

¹H NMR (500 MHz, CDCl₃) δ 0.8-1.9 (m, 10H), 1.95 (bd, 1H), 2.4-2.6 (m,4H), 2.7-2.8 (m, 3H; thereof 2.79 (d, 1H)), 4.13 (m, 1H), 4.37 (dd, 1H),4.60 (dd, 1H), 14.97 (dd, 1H), 5.09 (dd, 2H), 5.22 (s, 2H), 7.25-7.4 (m,10H), 7.47 (d, 2H), 7.83 (d, 2H), 8.61 (bt, 1H).

(iii) HOOC—CH₂—CH₂—(R)Cgl-Aze-Pab×2 HCl

BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pab(Z), 0.10 g (0.15 mmol), was dissolved in 10ml of ethanol and hydrogenated over 5% Pd/C at atmospheric pressure for1 h.

The solution was filtered, evaporated and the crude product was purifiedon RPLC using CH₃CN/0.1 M NH₄OAc (1/4). The resulting product was freezedried, treated with an excess of conc. HCl and freeze dried again togive 31 mg of the dihydrochloride salt.

¹H NMR (300 MHz, D₂O) δ 0.8-2.1 (m, 11H), 2.38 (m, 1H), 2.7-2.9 (m, 3H),3.2-3.4 (m, 2H), 3.98 (d, 1H), 4.35-4.55 (m, 2H), 4.60 (s, 2H), 5.04(dd, 1H), 7.59 (d, 2H), 7.83 (d, 2H).

¹³C NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.2, 167.8,172.3 and 175.5.

Example 3

HOOC—CH₂—(R)Cgl-Pro-Pab×2 HCl

(i) Boc-(R)Cgl-Pro-Pab(Z)

Boc-(R)Cgl-Pro-OH (See preparation of starting materials), 2.3 g (6.49mmol), DMAP, 2.38 g (19.47 mmol), and H-Pab(Z)(See preparation ofstarting materials), 1.84 g (6.49 mmol) was mixed in 30 ml acetonitrile.The mixture was cooled to −15“c and EDC, 1.31 g (6.81 mmole) was added.The temperature was allowed to reach room temperature and the mixturewas stirred over night. After evaporation, the residue was dissolved inethyl acetate and 0.3 M KHSO₄-solution. The acidic water phase wasextracted three times with ethyl acetate. The organic phase was washedtwice with a 0.3 M KHSO₄-solution, twice with a NaHCO₃-solution and oncewith brine, dried (Na₂SO₄), filtered and evaporated. The crude productwas purified by flash chromatography on silica gel using ethyl acetateas eluent to yield 1.77 g (44%) of the product.

¹H-NMR (500 MHz, CDCl₃): δ 0.9-1.49 (m, 14H), 1.5-2.1 (m, 9H), 2.37 (bs,1H), 3.53 (q, 1H), 3.94 (bs, 1H), 4.02 (m, 1H), 4.43 (bs, 2H), 4.65 (d,1H), 5.09 (bs, 1H), 5.20 (s, 2H), 7.18-7.4 (m, 5H), 7.45 (d, 2H), 7.62(bs, 1H), 7.81 (m, 2H),

(ii) H—(R)Cgl-Pro-Pab(Z)

1.45 q (2.34 mmol) of Boc-(R)Cgl-Pro-Pab(Z) was dissolved in 75 ml HClsaturated ethyl acetate. The mixture was allowed to stand for 10 min atroom temperature. The solvent was evaporated to yield 1.3 g of thedihydrochloride salt of the product.

¹H-NMR (300 MHz, D₂O): δ 1.0-1.45 (m, 5H), 1.58-2.2 (m, 9H), 2.3-2.5 (m,1H), 3.75-3.90 (m, 2H), 4.25 (d, 2H), 4.5-4.66 (m, 3H), 5.49 (s, 2H),7.45-7.7 (m, 7H), 7.87 (d, 2H)

The amine was obtained by dissolving the dihydrochloride salt in 0.1 MNaOH-solution and extracting the water phase three times with ethylacetate. The organic phase was washed once with brine, dried (Na₂SO₄),filtered and evaporated to yield 1.19 g (97%) of the title compound.

(iii) BnOOC—CH₂—(R)Cgl-Pro-Pab(Z)

0.340 g (0.65 mmole) H—(R)Cgl-Pro-Pab(Z) was mixed with 0.215 g (0.65mmole) BnOOC—CH₂—OTf (see preparation of starting materials), 0.299 g(2.17 mmole) K₂CO₃ in 4 ml dichloromethane and refluxed for half anhour. The reaction mixture was then stirred over night at roomtemperature. The reaction mixture was diluted with CH₂Cl₂ and theorganic layer was washed once with water and brine, dried (Na₂SO₄),filtered and evaporated. The crude product was purified by flashchromatography using a stepwise gradient of CH₂Cl₂/MeOH (97/3 followedby 95/5) to give 299 mg of a mixture of two products according to TLC.The mixture was therefore purified further by flash chromatography usinga stepwise gradient of ethyl acetate/toluene (9/1, 93/7, 95/5, 100/0) togive 46 mg (9%) of (BnOOC—CH₂)₂—(R)Cgl-Pro-Pab(Z) which eluated firstfrom the column followed by 133 mg (31%) of the desired productBnOOC—CH₂—(R)Cgl-Pro-Pab(Z).

¹H-NMR (300 MHz, CDCl₃): BnOOC—CH₂—(R)Cgl-Pro-Pab(Z): δ 0.9-1.3 (m, 5H),1.4-2.1 (m, 9H), 2.3-2.4 (m, 1H), 3.05 (d, 1H), 3.20-3.37 (AB-systemcentered at δ 3.29, 2H), 3.5-3.6 (m, 2H), 4.29-4.57 (ABX-system centeredat d 4.43, 2H), 4.62 (d, 1H), 4.91 (apparent singlet, 2H), 5.19 (s, 2H),6.75 (bs, NH), 7.1-7.5 (m, 12H), 8.7-8.8 (m, 2H+NH), 9.45 (bs, NH)

¹H-NMR (300 MHz, CDCl₃): (BnOOC—CH₂)₂—(R)Cgl-Pro-Pab(Z): δ0.68-0.9 (m,2H), 1.0-1.3 (m, 3H), 1.43 (bd, 1H), 1.55-2.0 (m, 7H), 2.05 (bd, 1H),2.3-2.4 (m, 1H), 3.15 (d, 1H), 3.25-3.48 (m, 2H), 3.55-3.79 (AB-systemcentered at d 3.67, 4H), 4.38-4.58 (ABX-system centered at d 4.48, 2H),4.68 (d, 1H), 4.82-4.98 (AB-system centered at d 4.91, 4H), 5.19 (s,2H), 6.66 (bs, NH), 7.1-7.5 (m, 17H), 7.75 (d, 2H), 7.80 (t, NH), 9.37(bs, NH)

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.7, 168.1,171.5, 172.3 and 172.6

(iv) HOOC—CH₂—(R)Cgl-Pro-Pab×2 HCl

0.133 g (0.20 mmole) of BnOOC—CH₂—(R)Cgl-Pro-Pab(Z) was mixed with 0.060g 5% Pd/C, 1 ml 1M HCl-solution and 10 ml ethanol. The mixture wastreated under H₂-atmosphere for one hour. After filtration through hyfloand evaporation of the solvent the product in 90% yield, 93 mg, wasobtained by freeze drying twice from water.

¹H-NMR (300 MHz, D₂O): δ 1.0-1.45 (m, 5H), 1.5-2.1 (m, 9H), 2.2-2.4 (m,1H), 3.55-3.85 (m, 4H; thereof 3.79 (s, 2H)), 4.23 (d, 1H), 4.33-4.57(m, 3H), 7.44 (d, 2H), 7.69 (d, 2H)

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 166.9, 167.2,169.1, 174.5

Example 4

HOOC—CH₂—CH₂—(R)Cgl-Pro-Pab×2 HCl

(i) BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pab(Z)

0.406 g (0.782 mmole) of H—(R)Cgl-Pro-Pab(Z) (See Example 3) wasdissolved in 3 ml ethanol and 132 μl (0.861 mmole) of bensylacrylate wasadded. The reaction mixture was stirred for three days at roomtemperature. The mixture was evaporated and the crude product purifiedby flash chromatography using a stepwise gradient of CH₂Cl₂:MeOH 95/5and 90/10 as eluent to yield 0.399 g (75%) of the product.

¹H-NMR (300 MHz, CDCl₃):δ 0.8-1.0 (m, 1H), 1.0-1.3 (m, 4H), 1.35-2.2 (m,9H), 2.3-2.6 (m, 4H), 2.65-2.78 (m, 1H), 3.05 (d, 1H), 3.4-3.6 (m, 2H),4.25-4.52 (ABX-system central at d 4.40, 2H), 4.64 (dd, 1H), 5.05 (s,2H), 5.20 (s, 2H), 7.2-7.38 (m, 10H), 7.43 (d, 2H), 7.78 (d, 2H)

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.7, 167.9,171.3, 172.7 and 175.4.

(ii) HOOC—CH₂—CH₂—(R)Cgl-Pro-Pab×2 HCl

0.261 g (0.383 mmole) of BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pab(Z) was mixed with0.075 g 5% Pd/C, 1 ml 1M HCl-solution and 10 ml ethanol. The mixture washydrogenated at atmospheric pressure for two hours. After filtrationthrough hyflo and evaporation of the solvent the product 0.196 g (96%)was obtained by freeze drying twice from water

¹H—NMR (300 MHz, D₂O): δ 1.17-1.40 (m, 5H), 1.60-1.92 (m, 5H), 1.92-2.2(m, 4H), 2.32-2.48 (m, 1H), 2.81 (t, 2H), 3.11-3.36 (ABX₂-systemcentered at δ 3.24, 2H), 3.63-3.90 (m, 2H), 4.25 (d, 1H), 4.42-4.63 (m,3H), 7.54 (d, 2H), 7.78 (d, 2H)

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.0, 167.30,174.6 and 174.7.

Example 5

(HOOC—CH₂)₂—(R)Cgl-Pro-Pab×2 HCl

46 mg (0.056 mmole) of (BnOOC—CH₂)₂—(R)Cgl-Pro-Pab(Z) (See Example 3)was mixed with 25 mg 5% Pd/C, 0.7 ml 1M HCl-solution and 7 ml ethanol.The mixture was hydrogenated at atmospheric pressure for one hour. Thecatalyst was filtered off through hyflo and the solvent evaporated. Thefinal product 25 mg (77%) was obtained by freeze drying twice fromwater.

¹H-NMR (300 MHz, D₂O): δ 1.0-1.4 (m, 5H), 1.45-2.2 (m, 9H), 2.25-2.45(m, 1H), 3.53-3.84 (m, 2H), 3.84-4.22 (AB-system centered at δ 4.03,4H), 4.26 (d, 1H), 4.35-4.6 (m, 3H), 7.53 (d, 2H), 7.77 (d, 2H)

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.1, 167.3,170.6 and 174.5

Example 6

H—(R)Cgl-Pic-Pab×2 HCl

(i) Boc-(R)Cgl-Pic-Pab(Z)

0.478 g (2.49 mmol) EDC was added at −18° C. to a stirred solution of0.875 g (2.37 mmol) Boc-(R)Cgl-Pic-OH (See preparation of startingmaterials), 1.22 g (9.97 mmol) DMAP, and 0.706 g (2.49 mmol) H-Pab(Z)(See preparation of starting materials) in a mixture of 30 mlacetonitrile and 1 ml DMF. The reaction mixture was allowed to reachroom temperature during a couple of hours and stirring was continued for48 h. The solvent was removed in vacuo and the residue was dissolved in50 ml ethyl acetate. The solution was washed with 15 ml water, 3×15 ml0.3 M KHSO₄, 2×15 ml Na₂CO₃ solution and water. Removal of the solventgave a residue which was subjected to flash chromatography using ethylacetate/heptane 9:1 as eluent. The yield was 0.96 g (64%).

(ii) H—(R)Cgl-Pic-Pab(Z)

Hydrogen chloride was bubbled through a solution of 0.56 g (0.88 mmol)Boc-(R)Cgl-Pic-Pab(Z) in 25 ml ethyl acetate. After a couple of minutescrystals precipitated from the solution. The solvent was removed invacuo and 50 ml ethyl acetate was added. Washing with 2×15 ml 2 M sodiumhydroxide solution and extraction of the aqueous phase with 25 ml ethylacetate was followed by drying (sodium sulphate) of the combinedextracts and removal of the solvent in vacuo to give 0.448 g (95%) ofthe desired product.

(iii) H—(R)Cgl-Pic-Pab×2 HCl

A solution of 98 mg (0.18 mmol) H-Cgl-Pic-Pab(Z) in 5 ml 95% ethanol and1 ml water was stirred in an atmosphere of hydrogen for 4 hours in thepresence of 5% Pd/C. The mixture was filtered and 0.3 ml 1 Mhydrochloric acid was added. The ethanol was removed in vacuo and theresidue was freeze dried to give 70 mg (81%) of the desired compound.

¹H-NMR (300 MHz, CD₃OD): δ 1.00-1.56 (m, 7H), 1.56-1.94 (m, 9H), 2.32(bd, 1H), 3.32-3.45 (m, 1H), 3.90 (bd, 1H), 4.35 (d, 1H), 4.50 (s, 2H),5.10-5.20 (m, 1H), 7.55 (d, 2H), 7.76 (d, 2H)

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.2, 170.5 and173.4.

Example 7

HOOC—CH₂—(R,S)CH(COOH)—(R)Cgl-Pic-Pab×2 HCl

(i) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cgl-Pic-Pab(Z)

A mixture of 350 mg (0.66 mmol) H—(R)Cgl-Pic-Pab(Z) (See Example 6) and233 mg dibenzyl maleate in 2.5 ml ethanol was kept at room temperaturefor 4 days. The ethanol was removed in vacuo and the residue wassubjected to flash chromatography using ethyl acetate/heptane 9:1 aseluent to give 0.108 mg of the product.

(ii) HOOC—CH₂—(R,S)CH(COOH)—(R)Cgl-Pic-Pab×2 HCl

105 mg (0.13=mmol) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cgl-Pic-Pab(Z) dissolvedin 5 ml 95% ethanol and 1 ml water was hydrogenated for 5 hours in thepresence of 5% Pd/C. 0.3 ml 1 M hydrochloric acid was added and themixture was filtered and the solvent was removed in vacuo. The residuewas dissolved in water and freeze dryed to yield 54 mg (73%) of thedesired substance.

¹H-NMR (300 MHz, CD₃OD, mixture of two diastereomers 5/4): δ 1.10-1.60(m, 7H), 1.60-2.04 (m, 9H), 2.23-2.42 (m, 1H), 2.93-3.15 (m 2H),3.30-3.42 (m, 1H, partially hidden by the MeOD-peak), 3.71-3.95 (m, 1H),3.98-4.10 (m, 1H), 4.40-4.60 (m, 3H), 5.10-5.20 (m, 1H), 7.49-7.60 (m,2H), 7.70-7.81 (m, 2H)

¹³C-NMR (75 MHz D₂O): amidine and carbonyl carbons: δ 167.1, 168.95,169.6 and 173.1.

MS m/z 516 (M⁺+1)

Example 8

H—(R)Cha-Aze-Pab×2 HCl

(i) Boc-(R)Cha-Aze-Pab(Z)

409 mg (2.13 mmol) EDC was added at −18° C. to a stirred mixture of 0.72g (2.03 mmol) Boc-(R)Cha-Aze-OH (See preparation of starting materials),1.04 g (8.53 mmol) DMAP, and 604 mg (2.13 mmol) H-Pab(Z) (Seepreparation of starting materials) in 20 ml acetonitrile. The reactionmixture was allowed to reach room temperature over night and the solventwas subsequently removed in vacuo. The residue was dissolved in 40 mlethyl acetate and the organic phase was washed succesively with 10 mlwater, 3×10 ml 0.3M KHSO4, 2×10 ml Na₂CO₃—NaCl (aq), and finally 10 mlBrine. Drying (Na₂SO₄) and removal of the solvent in vacuo gave aresidue which was subjected to flash chromatography using ethylacetate/methanol 9:1 as eluent to yield 645 mg (51%) of the titlecompound.

(ii) H—(R)Cha-Aze-Pab(Z)

Hydrogen chloride was bubbled through a solution of 640 mg (1.03 mmol)Boc-(R)Cha-Aze-Pab(Z) in 25 ml of ethyl acetate. After a couple ofminutes, TLC analysis indicated the completion of the reaction. Vacuumwas applied to remove excess hydrogen chloride and the mixture was thendiluted to 50 ml with ethyl acteate. Washing with 2×15 ml Na₂CO₃ (aq)was followed by extraction of the aqueous phase with 15 ml ethylacetate. The combined organic extracts were washed with water and dried(Na₂CO₃) and the solvent was removed in vacuo to give 513 mg (96%) ofH—(R)Cha-Aze-Pab(Z).

(iii) H—(R)Cha-Aze-Pab×2 HCl

76 mg (0.15 mmol) H—(R)Cha-Aze-Pab(Z) dissolved in 5 ml 95% ethanol and1 ml water was hydrogenated at atmospheric pressure in the presence of5% Pd/C for 4 h. Removal of the catalyst by filtration, addition of 0.4ml 1M hydrochloric acid and evaporation of the solvent in vacuo gave aresidue which was dissolved in 2 ml water. Freeze drying gave 57 mg(85%) of the product.

¹H-NMR (500 MHz, D₂O, 2 rotamers, 3:1 mixture): δ 1.02-1.20 (m, 2H),1.22-1.92 (m, 11H), 2.40-2.50 (m, 1H), 2.80-2.90 (m, 1H), 4.25 (bt, 1H),4.40 (dq, 1H), 4.53 (dq, 1H), 4.65 (s, 2H), 5.05-5.10 (m, 1H), 7.65 (d,2H), 7.88 (d, 2H).

Chemical shifts of resolved signals of the minor rotamer: δ 0.57 (m),0.85 (m), 2.95 (m), 4.06 (dq), 4.17 (dq), 4.63 (s), 5.33 (m), 7.70 (d),7.93 (d).

¹³C-NMR (125 MHz D₂O): amidine and carbonyl carbons: δ 167.2, 170.4 and172.8.

Example 9

HOOC—CH₂—(R)Cha-Aze-Pab×2 HCl

(i) BnOOC—CH₂—(R)Cha-Aze-Pab(Z)

0.119 g (0.52 mmol) benzyl bromoacetate was added to a mixture of 0.27 g(0.52 mmol) H—(R)Cha-Aze-Pab(Z) (See Example 8) and 0.158 g (1.14 mmol)K₂CO₃ in 5.2 ml acetonitrile and heated to 60° C. in an oilbath for 1 h.The solvent was removed and ethyl acetate and water was added. Thephases were separated and the organic phase was washed with brine anddried (Na₂SO₄). Evaporation in vacuo gave 0.344 g of a residue which wassubjected to flash chromatography using ethyl acetate as eluent, andthen another time using ethylacetate:tetrahydrofuran: NH₃-saturatedmethanol (60:5:2) to give 0.163 g of the desired product.

¹H-NMR (300 MHz, CDCl₃); δ 0.7-1.0 (m, 2H). 1.05-2.05 (m, 11H),2.35-2.55 (m, 1H), 2.55-2.75 (m, 1H), 3.15-3.32 (m, 3H), 3.95-4.05 (t,2H), 4.4 and 4.5 (ABX-system, 2H), 4.8-4.95 (m, 1H), 5.05 (s, 2H), 5.2(s, 2H), 7.2-7.5 (m, 12H), 7.7-7.85 (d, 2H), 8.3-8.45 (t, 1H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 167.8,170.7, 171.9 and 175.9.

(ii) HOOC—CH₂—(R)Cha-Aze-Pab×2 HCl

0.163 g (0.243 mmol) BnOOC—CH₂—(R)Cha-Aze-Pab(Z) dissolved in 5.5 mlethanol (99.5%) and 0.7 ml hydrogen chloride (1 N) was hydrogenated inthe presence of 0.17 g 5% Pd/C for 4 h. Removal of the catalyst byfiltration and evaporation of the solvent followed by dissolving inwater and freeze drying gave 107 mg (85%) of the title compound.

¹H-NMR (500 MHz, CD₃OD, mixture of two rotamers): major rotamer: δ0.95-1.95 (m, 13H), 2.3-2.4 (m, 1H), 2.6-2.75 (m, 1H), 3.5-3.75 (m, 2H),4.05-4.15 (m, 1H), 4.15-4.23 (m, 1H), 4.36-4.43 (m, 1H), 4.43-4.5 (m,1H), 4.58-4.65 (m, 1H), 4.83-4.88 (m, 1H), 7.5-7.6 (m, 2H), 7.73-7.82(m, 2H).

Resolved signals from the minor rotamer appears at δ 2.2-2.3 (m),3.95-4.05 (m), 5.1-5.17 (m), 7.6-7.67 (m).

¹³C-NMR (75 MHz, CD₃OD): amidine and carbonyl carbons; δ 168.2, 169.8,168.9 and 172.3.

Example 10

HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Aze-Pab×2 HCl

(i) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Aze-Pab(Z)

A mixture of 230 mg (0.443 mmol) H—(R)Cha-Aze-Pab(Z) (See Example 8) and144 mg (0.487 mmol) dibenzyl maleate in 1.5 ml 95% ethanol was stirredat ambient temperature for 5 days. After removal of the ethanol invacuo, the residue was subjected to flash chomatography using ethylacetate/methanol 95/5 as eluent to give 54 mg (15%) of the product.

(ii) HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Aze-Pab

49 mg (0.06 mmol) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Aze-Pab(Z) dissolvedin 5 ml 95% ethanol and 1 ml water was hydrogenated in the presence of5% Pd/C for 4.5 h. Removal of the catalyst by filtration and evaporationof the solvent in vacuo gave a residue which was dissolved in 2 ml waterand 0.2 ml 1M hydrochloric acid. Freeze drying gave 32 mg (93%) of theproduct.

The ¹H-NMR spectrum of the title compound in D₂O exhibits two sets ofstrongly overlapping signals arising from the two diastereomers.Additionally resolved resonances of a minor rotamer, integrating toapproximatly 15% also appears in the spectrum.

¹H-NMR (300 MHz, D₂O): δ 1.03-2.00 (m, 13H), 2.32-2.53 (m, 1H),2.72-2.96 (m_(,) 1H), 3.06-3.28 (m, 2H), 4.10-4.55 (m, 4H), 4.62 (bs,2H), 5.00-5.10 (m, 1H), 7.55-7.68 (m, 2H), 7.80-7.94 (m, 2H)

Resolved signals from the minor rotamer appears at δ 0.65 (m), 0.80 (m),4.00 (m), 5.24 (m), 5.35 (m).

¹³C-NMR (75 MHz D₂O): amidine and carbonyl carbons: δ 167.2, 169.0,171.0, 172.3 and 174.1.

Example 11

HOOC—CH₂—(R or S)CH(COOH)-Cha-Aze-Pab/a×2 HCl

(i) BnOOC—CH₂—(R or S)CH(COOBn)—(R)Cha-Aze-Pab(Z)/a

A mixture of 2.0 g (3.8491 mmol) H—(R)Cha-Aze-Pab(Z) (See Example 8) and1.37 g dibenzyl maleate in 10 ml 95% ethanol was stirred at ambienttemperature for 4 days. After removal of the ethanol in vacuo, theresidue was subjected to flash chromatography using ethylacetate/methanol 98/2 as eluent to give 1.024 g (32%) ofBnOOC—CH₂—(R,S)CH(COOBn)-(R)Cha-Aze-Pab(Z). The two diastereomers wereseparated by RPLC using (CH₃CN/0.1 M NH₄OAc 65/35) as eluent. Thisdiastereomer eluted first from the column. After removal of theacetonitrile in vacuo the water phase was extracted three times withethyl acetate. The organic phase was washed once with water dried(Na₂SO₄), filtered and evaporated to yield 0.352 g of the title compoundas a pure stereoisomer.

(ii) HOOC—CH2—(R or S)CH(COOH)—(R)Cha-Aze-Pab/a 2×HCl 350 mg (0.43 mmol)BnOOC—CH₂—(R or S)CH(COOBn)—(R)Cha-Aze-Pab(Z)/a (The diaststereomer from(i) above) dissolved in 15 ml 95% ethanol and 3 ml water washydrogenated in the presence of 5% Pd/C for 4.5 h. Removal of thecatalyst by filtration and evaporation of the solvent in vacuo gave aresidue which was dissolved in 5 ml water and 1.0 ml 1M hydrochloricacid. Freeze drying gave 214 mg (87%) of the product as a purestereoisomer.

¹H-NMR (300 MHz, MeOD, mixture of two rotamers): δ 0.85-1.93 (m, 13H),2.25-2.38 (m, 1H), 2.60-2.75 (m, 1H), 2.88 (dd, 2H), 3.92 (t, 1H),4.15-4.25 (m, 2H), 4.30-4.43 (m, 1H), 4.56 (AB-system, 2H), 4.76-4.86(m, 1H, partially obscured by the solvent signal), 7.59 (d, 2H), 7.78(d, 2H).

Resolved signals arising from the minor rotamer appears at δ 0.70, 2.95,3.82, 4.00, 5.08 and 7.83

¹³C-NMR (75 MHz D₂O): amidine and carbonyl carbons: δ 166.9, 168.8,171.7, 172.3 and 173.8.

Example 12

HOOC—CH₂—(RorS)CH(COOH)—(R)Cha-Aze-Pab/b×2 HCl

(i) BnOOC—CH₂—(R or S)CH(COOBn)—(R)Cha-Aze-Pab(Z)/b

The title compound was obtained by using the same procedure as describedin Example 11 above on BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Aze-Pab(Z). Thisdiastereomer came out after the first one from the column. Yield 0.537g.

(ii)HOOC—CH₂—(RorS)CH(COOH)—(R)Cha-Aze-Pab/b×2 HCl

530 mg (0.65 mmol) BnOOC—CH₂—(R or S)CH(COOBn)—(R)Cha-Aze-Pab(Z)/bdissolved in 15 ml 95% ethanol and 3 ml water was hydrogenated in thepresence of 5% Pd/C for 5 h. Removal of the catalyst by filtration andevaporation of the solvent in vacuo gave a residue which was dissolvedin 6 ml water and 1.0 ml 1M hydrochloric acid. Freeze drying gave 290 mg(78%) of the product.

¹H-NMR (300 MHz, MeOD, mixture of two rotamers): δ 0.86-1.90 (m, 13H),2.30-2.42 (m, 1H), 2.60-2.75 (m,1H), 2.75-2.85 (m, 1H), 2.95-3.05 (m,1H), 3.65-3.71 (m, 1H), 4.00-4.10 (m, 1H), 4.14-4.24 (m, 1H), 4.36-4.62(m, 3H), 4.78-4.86 (m, 1H partially obscured by the solvent signal),7.57 (d, 2H), 7.75 (d, 2H).

Resolved signals arising from a minor rotamer appears at δ 0.78, 2.92,3.82, 5.36 and 7.80

¹³C-NMR (75 MHz D₂O): amidine and carbonyl carbons: δ 166.8, 169.0,172.0, 172.4 and 175.2.

Example 13

HOOC—CH₂—CH₂—(R)Cha-Aze-Pab×2 HCl

(i) BnOOC—CH₂—CH₂—(R)Cha-Aze-Pab(Z)

A mixture of 182 mg (0.35 mmol) H—(R)Cha-Aze-Pab(Z) (See Example 8) and62.5 mg (0.385 mmol) benzyl acrylate in 1.5 ml 95% ethanol was stirredat room temperature for 4 days. The solvent was removed in vacuo and theresidue was subjected to flash chromatography using ethylacetate/methanol 9:1 as eluent to give 200 mg (84%) of the titlecompound.

(ii) HOOC—CH₂—CH₂—(R)Cha-Aze-Pab×2 HCl

195 mg (0.29 mmol) BnOOC—CH₂—CH₂—(R)Cha-Aze-Pab(Z) dissolved in 10 ml95% ethanol and 2 ml water was hydrogenated in the presence of 5% Pd/Cfor 4 h. Removal of the catalyst by filtration and evaporation of thesolvent in vacuo gave a residue which was dissolved in 2 ml water and0.4 ml 1M hydrochloric acid. Freeze drying gave 130 mg (86%) of theproduct.

¹H-NMR (500 MHz, CD₃OD): δ 0.98-1.27 (m, 2H), 1.30-1.90 (m, 11H),2.27-2.35 (m, 1H), 2.65-2.74 (m, 1H), 2.77 (t, 2H), 3.32 (t, 2H), 4.10(t, 1H), 4.17-4.25 (m, 1H), 4.40-4.49 (m, 1H), 4.55 (AB, 2H), 4.83-4.90(m, 1H), 7.58 (d, 2H), 7.77 (d, 2H).

¹³C-NMR (125 MHz D₂O): amidine and carbonyl carbons: δ 167.0, 168.9,172.4 and 174.6.

Example 14

HOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab×2 HCl

(i) BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab(Z)

A mixture of 0.212 g (0.408 mmole) H—(R)Cha-Aze-Pab(Z) (See Example 8),0.124 g (0.89 mmole) K₂CO₃ and 0.128 g (0.449 mmole)BnOOC—CH₂—NH—CO—CH₂—Br (See preparation of starting materials) in 6 mlacetonitrile was stirred at 50° C. for two hours. After evaporation ofthe solvent the residue was dissolved in water and ethyl acetate. Thewater layer was extracted twice with ethyl acetate and the combinedorganic layer was dried (Na₂SO₄), filtered and evaporated. The productwas purified by flash chromatography using a stepwise gradient of ethylacetate/tetrahydrofurane (85/15, 4/1, 7/3) to yield 0.190 g (64%) of thetitle compound.

¹H-NMR (300 MHz, CDCl₃): δ 0.75-2.1 (m, 13H), 2.43 (m, 1H), 2.56 (d,1H), 2.79 (m, 1H), 3.0-3.15 (m, 2H; thereof 3.05 (d, 1H)), 3.89-4.18 (m,5H), 4.8-4.98 (m, 2H), 5.15 (s, 2H), 5.18 (s, 2H), 7.2-7.47 (m, 12H),7.72 (t, NH), 7.86 (d, 2H), 8.14 (bs, NH), 8.31 (dd, NH), 9.42 (bs, NH)

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 168.7,169.22, 169.83, 171.7, 175.5

(ii) HOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab×2 HCl

0.19 g (0.26 mmole) of BnOOC-CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab(Z) was mixedwith 0.075 g 5 t Pd/C, 1.5 ml 1N HCl-solution, 3 ml water and 17 mlethanol and the mixture was hydrogenated at atmospheric pressure for onehour. Filtration of the catalyst, evaporation of the solvent followed byfreeze drying from water gave 144 mg (97%) of the title compound.

¹H-NMR (D₂O, 300 MHz, two rotamers 4:1): δ 0.88-1.88 (m, 13H), 2.25-2.42(m, 1H), 2.63-2.89 (m, 1H), 3.94 (s, 2H), 3.99 (apparent doublet, 2H),4.16 (t, 1H), 4.28 (q, 1H), 4.41 (q, 1H), 4.56 (s, 2H), 4.98 (dd, 1H),7.53 (d, 2H), 7.77 (d, 2H),

Resolved signals from the minor rotamer appears at δ 0.50 (bq), 0.77(bq), 5.21 (dd), 7.56 (d) and 7.81 (d).

¹³C-NMR (D₂O, 75 MHz): The carbonyls and amidinecarbon at δ 166.8,166.9, 168.6, 172.3 and 173.4.

Resolved signals from the minor rotamer appears at δ: 166.6, 169.6 and172.0

Example 15

H—(R)Cha-Pro-Pab×2 KCl

(i) Boc-(R)Cha-Pro-Pab(Z)

0.135 ml (1.1 mmol) pivaloyl chloride was added at room temperature to astirred mixture of 0.155 ml (1.1 mmol) triethyl amine and 405 mg (1.1mmol) Boc-(R)Cha-Pro-OH (See preparation of starting materials) in 5 mlDMF. After 3 h 340 mg (1.1 mmol) H-Pab(Z)(See preparation of startingmaterials) in 5 ml DMF was added and stirring was continued over night.The reaction mixture was diluted with water and extracted with ethylacetate/toluene 1:1. The organic phase was dried (MgSO₄) and the solventwas removed in vacuo to give a residue which was subjected to flashchromatography using ethyl acetate as eluent. The yield was 309 mg(49%).

(ii) H—(R)Cha-Pro-Pab(Z)

Hydrogen chloride was bubbled through a solution, until saturation, of1.246 g (2 mmol) Boc-(R)Cha-Pro-Pab(Z) in 20 ml ethyl acetate at roomtemperature. After 30 minutes sodium carbonate solution (10%) was addedand the organic phase which separated was dried (K₂CO₃). The dryingagent was washed with methylene chloride and the solvent was evaporatedfrom the combined organic phases to give 1.11 is g (100%) of the titlecompound.

(iii) H—(R)Cha-Pro-Pab×2 HCl

100 mg (0.19 mmol) H—(R)Cha-Pro-Pab(Z) dissolved in 15 ml ethanol washydrogenated in the presence of 38 mg 10% Pd/C for 1.5 h. Dilution ofthe reaction mixture with distilled water and removal of the catalyst byfiltration followed by removal of the ethanol in vacuo and freeze dryinggave the title compound as a colorless powder. The peptide was finallyconverted to the dihydrochloride by dissolution in hydrochloric acidfollowed by freeze drying to give 90 mg (100%) of the title compound.

¹H-NMR (300 MHz, D2O); & 1.0-2.0 (m, 13H), 2.0-2.3 (m, 3H), 2.3-2.5 (m,1H), 3.6-3.7 (m, 1H), 3.8-3.9 (m, 1H), 4.3-4.5 (t, 1H), 4.5-4.6 (m, 3H),7.4-7.6 (m, 3H), 7.6-7.9 (m, 2H).

¹³C-NMR (75 MHz, D2O): amidine and carbonyl carbons: δ 167.2, 170.0,174.9.

Example 16

HOOC—CH₂—(R)Cha-Pro-Pab×2 HCl

(i) BnOOC—CH₂—(R)Cha-Pro-Pab(Z)

A mixture of 268 mg (0.5 mmol) H—(R)Cha-Pro-Pab(Z) (See Example 15), 90μl (0.55 mmol) benzyl bromoacetate and 181 mg (1.3 mmol) K₂CO₃ in 2 mlacetonitrile was sonicated at 40° C. for 2.5 h. The mixture was filteredthrough hyflo and the solvent was removed in vacuo to give a residuewhich was subjected to flash chromatography using ethyl acetate aseluent to give 194 mg (57%) of the title compound.

HOOC—CH₂—(R)Cha-Pro-Pab×2 HCl

194 mg (0.28 mmol) BnOOC—CH₂—(R)Cha-Pro-Pab(Z) dissolved in 10 mlethanol was hydrogenated in the presence of 77 mg 10% Pd on charcoal for3 h. The reaction mixture was diluted with water and the catalyst wasremoved by filtration. Evaporation of the ethanol in vacuo followed byfreeze drying gave a white residue. Hydrochloric acid was added and theresulting solution was finally freeze dried to give 115 (68%) of thedesired product.

¹H-NMR (300 MHz, D₂O); δ 1.0-1.2 (m, 2H), 1.2-1.5 (m, 3H), 1.5-2.0 (m,5H), 2.0-2.3 (m, 3H), 2.3-2.5 (m, 1H), 3.6-3.8 (m, 1H), 3.8-4.0 (m, 3H),4.4-4.7 (m, 4H), 7.5-7.7 (d, 2H), 7.7-7.9 (d, 2H).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.1, 168.2,169.3, 174.6.

Example 17

HOOC—CH₂—(Me)(R)Cha-Pro-Pab

(i) Boc-(Me)(R)Cha-Pro-Pab(Z)

To a solution of 0.8 g (1.67 mmol) of Boc-(Me)(R)Cha-Pro-OSu (Seepreparation of starting materials) in 3 ml DMF was added a solution of0.562 g (1.85 mmol) of H-Pab(Z) (See preparation of starting materials)in 3 ml of DMF, and the pH of the resulting solution was adjusted to 8-9with N-methylmorpholine, whereafter the solution was stirred at roomtemperature for 2 days.

The solution was poured onto water, and the resulting mixture wasextracted with 3×25 ml of ethyl acetate. The organic solution was washedwith 1M KHSO₄ solution, 10% NaHCO₃ solution, water and brine, and dried(Na₂SO₄). Evaporation of the solvent gave 0.65 g (60%) of the titlecompound as a yellowish white powder.

(ii) Me—(R)Cha-Pro-Pab(Z)

A solution of 0.60 g (0.92 mmol) of Boc-(Me)(R)Cha-Pro-Pab(Z) in 50 mlof EtOH was saturated with HCl at 0° C., and the solution was stored inrefrigerator overnight. The resulting solution was evaporated todryness, and the residue was dissolved in a Na₂CO₃ solution, extractedwith 3×25 ml ethyl acetate. The extract was washed with brine andevaporated to give 0.4 g (791) of the compound as a white fluffy powder.

¹H-NMR (CDCl₃, 300 MHz): δ 0.8-1.0 (m, 2H), 1.1-1.4 (m, 5H), 1.4-1.55(m, 1H), 1.6-1.9 (m, 10H), 1.9-2.05 (m, 2H), 2.05-2.2 (m, 2H), 2,19(s,3H), 2.4-2.5 (m, 1H), 3.28 (dd, 1H), 3.41 (q, 1H), 3.62 (m, 1H), 4.42(m, 2H), 4.61 (d, 1H), 5.2 (s, 2H), 7.2-7.45 (m, 7H), 7.72 (t, 1H), 7.79(d, 2H).

(iii) BnOOC—CH₂-(Me)(R)Cha-Pro-Pab(Z)

A mixture of 0.40 g (0.73 mmol) of Me—(R)Cha-Pro-Pab(Z), 0.17 gBnOOC—CH₂Br and 0.20 g (2 equiv.) of K₂CO₃ (mortared) in 15 ml of CH₃CNwas stirred at room temperature overnight. The resulting mixture wasevaporated, ethyl acetate was added, and the mixture was washed withwater and brine, dried (Na2SO4), and evaporated. The crude product (0.69g) was subjected to flash chromatography (CH₂Cl₂/MeOH 10/1) yielding0.39 g (77%) of a light yellow very viscous oil.

HOOC—CH₂-(Me)(R)Cha-Pro-Pab

To a solution of 0.39 g (0.56 mmol) of BnOOC—CH₂-(Me)(R)Cha-Pro-Pab(Z)in 30 ml of EtOH was added 0.1 g of Pd/C (10%), and the substance washydrogenated at atmospheric pressure. The solution was filtered andevaporated, whereafter the remaining syrupy material was freeze dried toyield 0.25 g (95%) of the compund as a white crystalline powder.

¹H-NMR (300 MHz, CD₃OD): δ 0.85-1.1 (m, 2H), 1.1-1.4 (m, 6H), 1.5-1.85(m, 9H), 1.9-2.05 (m, 3H), 2.05-2.15 (m, 1H), 2.15-2.3 (m, 1H), 2.57 (s,3H), 3.32 (d, 1H), 3.55-3.75 (m, 2H), 3.95-4.1 (m, 2H), 4.35-4.5 (m,3H), 7.55 (d, 2H), 7.72 (d, 2H).

¹³C-NMR (75 MHz, CD₃OD): amidine and carbonyl carbons: δ 168.4, 171.5,174.7, 175.1.

Example 18

HOOC—CH₂—CH₂—(R)Cha-Pro-Pab×2 HCl

(i) BnOOC—CH₂—CH₂—(R)Cha-Pro-Pab(Z)

A mixture of 149 mg (0.28 mmol) H—(R)Cha-Pro-Pab(Z) (See Example 15) and66 mg (0.4 mmol) benzyl acrylate in 1.5 ml ethanol was kept at roomtemperature for 36 h. The solvent was removed in vacuo and the residuewas subjected to flash chromatography using ethyl acetate as eluent togive 124 mg (64%) of the desired product.

(ii) HOOC—CH₂—CH₂—(R)Cha-Pro-Pab×2 HCl

124 mg (0.18 mmol) BnOOC—CH₂—CH₂—(R)Cha-Pro-Pab(Z) dissolved in 10 mlethanol was hydrogenated for 1 h in the presence of 55 mg 10% Pd/C. Thecatalyst was removed by filtration and the solvent was removed in vacuo.The residue was dissolved in hydrochloric acid and the resultingsolution was freeze dried to give 87 mg (79%) of the title compound.

¹H-NMR (300 MHz, D₂O): δ 1.0-2.0 (m, 13H), 2.0-2.2 (m, 3H), 2.2-2.4 (m,1H), 2.7-2.8 (t, 2H), 3.2-3.3 (m, 1H), 3.3-3.4 (m, 1H), 3.5-3.7 (m, 1H),3.7-3.9 (m, 1H), 4.3-4.6 (m, 4H), 7.4-7.6 (m, 2H), 7.7.6-7.8 (m, 2H).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.0, 168.3 and174.6 (Two carbons are overlapping).

Example 19

HOOC—CH₂—CH₂—(Me)(R)Cha-Pro-Pab×2 HCl

(i) BnOOC—CH₂—CH₂-(Me)(R)Cha-Pro-Pab(Z)

To a solution of 274 mg (0.5 mmol) of Me—(R)Cha-Pro-Pab(Z) (See Example17) in 5 ml of EtOH (99%) was added 97.3 mg (0.6 mmol) of benzylacrylate and the reaction was stirred at room temperature. After 72 h anadditional amount of 16.2 mg (0.1 mmol) of benzyl acrylate was added andthe stirring continued for 24 h. The solvent was evaporated and theresidue was subjected to flash chromatography(CH₂Cl₂/MeOH(NH₃-saturated), 95/5) to give 198 mg (56%) of the titlecompound.

¹H-NMR (500 MHz, CDCl₃): δ 0.8-2.0 (several m, 16H), 2.14 (s, 3H),2.24-2.33 (m, 2H), 2.38-2.46 (m, 1H), 2.67 (t, 2H), 3.32-3.40 (m, 2H),3.71 (m, 1H), 4.36-4.44 (m, 2H), 4.58 (m, 1H), 5.03 (apparent s, 2H),5.20 (s, 2H), 7.25-7.37 (m, 10H), 7.43 (d, 2H), 7.64 (t, 1H(NH)), 7.81(d, 2H).

¹³C-NMR (125 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.7, 167.9,171.7, 172.3 and 172.6.

(ii) HOOC—CH₂—CH₂-(Me)(R)Cha-Pro-Pab×2 HCl

To a solution of 198 mg (0.27 mmol) BnOOC—CH₂—CH₂-(Me)(R)Cha-Pro-Pab(Z)in 10 ml EtOH and 1 ml 1M HCl was added 60 mg of 5% Pd/C (containg 50%H₂O by weight) and the mixture was hydrogenated at athmospheric pressurefor 4 h. The catalyst was filtered off and the solvent was evaporated.The remaining oil was dissolved in water and freeze dried to give thetitle compound in a quantitative yield.

¹H-NMR (500 MHz, D₂O): δ 1.08-1.2 (m, 2H), 1.2-1.42 (m, 4H), 1.68-1.91(m, 5H), 1.93-2.08 (m, 2H), 2.09-2.26 (m, 3H), 2.49 (m, 1H), 2.95 (m,2H), 3.03 (s, 3H), 3.60 (apparent bs, 2H), 3.82 (m, 1H), 3.98 (m, 1H),4.53 (m, 1H), 4.61 (bs, 2H), 4.64 (m, 1H), 7.63 (d, 2H), 7.97 (d, 2H).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.2, 167.8 and174.5. Two peaks are probably overlapping.

Example 20

HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pro-Pab/a×2 HCl

(i) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pro-Pab(Z)

A mixture of 0.50 g (0.94 mmol) of H—(R)Cha-Pro-Pab(Z) (See Example 15)and 0.28 q (0.94=mol) of dibenzyl maleate in 20 ml of EtOH was kept atroom temperature for 5 days. Evaporation of the solvent followed byflash chromatography using CH₂Cl₂/MeOH as eluent gave 0.15 g (19%) ofthe diastereomeric mixture.

¹H NMR (500 MHz, CDCl₃) δ 0.7-2.1 (m, 17H), 2.3-2.4 (m, 1H), 2.5-2.8 (m,2H), 3.2-3.7 (in, 4H), 4.46 (d, 1H), 4.65 (bd, 1H), 4.81 (d, 1H),4.9-5.1 (m, 3H), 5.20 (s, 2H), 7.1-7.4 (m, 15H), 7.4-7.5 (m, 2H),7.6-7.8 (m, 3H).

(ii) HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pro-Pab/a×2 HCl

A mixture of 0.15 g (0.18 mmol) ofBnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pro-Pab(Z) was dissolved in 5 ml ofethanol and was hydrogenated over 5% Pd/C at atmospheric pressure for 1h. to give HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Pro-Pab. The two diastereomerswere separated by RPLC using (CH₃CN/0.1 M NH₄OAc 15/85) as eluentfollowed by freeze drying from HCl. This diastereomer eluted first fromthe column. Yield 19 mg (18%).

¹H-NMR (500 MHz, D₂O, mixture of two rotamers) major rotamer: δ 1.0-2.0(m, 15H), 2.15 (m, 2H), 2.44 (m, 1H), 3.00 (bd, 1H), 3.05 (bd, 1H), 3.69(m, 1H), 3.84 (m, 1H), 3.97 (bs, 1H), 4.5-4.7 (1,3H), 7.62 (d, 2H), 7.87(d, 2H).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.2, 168.3,173.8, 174.6 and 178.2.

Example 21

HOOC—C₂—(RorS)CH(COOH)—(R)Cha-Pro-Pab/b×2 HCl

The title compound was obtained by using the same procedure as describedin Example 20 an HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Pro-Pab. Thisdiastereomer came out after the first one from the column. Yield 19 mg(18%).

¹H-NM (500 MHz, D₂O, mixture of two rotamers) major rotamer: δ 1.0-2.0(m, 14H), 2.15-2.25 (m, 3H), 2.44 (m, 1H), 3.11 (bd, 1H), 3.19 (bd, 1H),3.71 (m, 1H), 3.92 (m, 1H), 4.03 (bs, 1H), 4.5-4.7 (m, 3H), 7.58 (d,2H), 7.84 (d, 2H).

Resolved signals arising from the minor rotamer appears at: δ 7.66 (d)and 7.91 (d).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.3, 168.5 and174.7. Two carbons are probably overlapping.

Example 22

HOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab×2 KCl

(i) BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab(Z)

0.246 g (0.460 mmole) of H—(R)Cha-Pro-Pab(Z) (See Example 15), 0.140 g(1.01 mmole) K₂CO₃ and 0.145 g (0.506 mmole) BnOOC—CH₂—NH—CO—CH₂—Br (Seepreparation of starting materials) was mixed in 6 ml acetonitrile. Themixture was stirred at 50° C. for 2 h 30 minutes, the solvent wasevaporated and the residue was partitioned between water and ethylacetate. The layers were separated and the water layer was extracted onemore time with ethyl acetate. The combined organic layer was dried(Na₂SO₄), filtered and evaporated to yield 0.350 g of an oil. The crudeproduct was purified by flash chromatography using a stepwise gradientof CH₂Cl₂/MeOH 97/3, 95/5, 92.5/7.5 to yield 0.227 g (67%) of the titlecompound.

¹³C-NMR (75 MHz, CDCl₃): δ 25.0, 26.0, 26.2, 26.4, 26.7, 32.4, 34.2,34.4, 40.8, 40.9, 42.9, 46.7, 50.5, 58.4, 60.2, 67.0, 67.2, 127.5,127.8, 128.2, 128.3, 128.4, 128.5, 128.6, 128.6, 134.1, 135.2, 137.0,142.6, 164.7, 168.9, 169.3, 170.4, 172.2, 175.0

(ii) HOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab×2 HCl

0.089 g (0.12 mmole) BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab(Z) was mixedwith 30 mg 5% Pd/C and dissolved in 10 ml acetic acid. The mixture washydrogenated at athmospheric pressure for one and a half hour.Filtration of the catalyst through hyflo and freeze drying with 1 ml 1Nhydrochloric acid gave 0.058 g (82%) of the desired product.

¹H—NMR (300 MHz, D₂O): δ 0.9-2.2 (m, 16H), 2.25-2.47 (m, 1H), 3.55-3.7(m, 1H), 3.7-4.1 (m, 5H), 4.42 (t, 1H), 4.48-4.6 (m, 3H), 7.51 (d, 2H),7.77 (d, 2H)

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 166.8, 167.1,168.2, 173.6 and 174.6

Example 23

EtOOC—CH₂—CH₂—CH₂—(R)Cha-Pro-Pab×HOAc

(i) EtOOC—CH═CH—CH₂—(R)Cha-Pro-Pab(Z)

H—(R)Cha-Pro-Pab(Z) (See Example 15) (275 mg, 0.51 mmol) was treatedwith K₂CO₃ (141 mg, 1.02 mmol) and BrCH₂CH═CHCOOEt (108 mg, 0.56 mmol)in CH₃CN (10 ml) at 20° C. for 20 h. The solvent was evaporated and theresidue was dissolved in EtOAc (5 ml)/H₂O (2 ml). The organic layer wasseparated, dried (Na₂SO₄), and concentrated yielding 397 mg of an oilwhich was purified by flash chromatography using EtOAc/Heptane, 1/4 aseluent to give 252 mg (77%) of the title compound.

¹H-NMR (500 MHz, CDCl₃): δ 0.8-1.05 (m, 2H), 1.1-1.45 (m, 3H), 1.3 (t,3H), 1.5-1.9 (m, 8H), 1.95-2.05 (m, 1H), 2.1-2.15 (m, 1H), 2.45-2.55 (m,1H), 3.0 and 3.15 (two d, 2H), 3.35-3.45 (m, 2H), 3.55-3.65 (m, 1H),4.15 (q, 2H), 4.3 (d, 1H), 4.6-4.7 (m, 2H), 5.2 (s,2H), 5.85 (d, 1H),6.75 (dt, 1H), 5.3-5.4 (m, 4H), 7.45 (d, 2H), 7.85 (d, 2H).

¹³C-NMR (75.0 MHz, CDCl₃): amidine and carbonyl carbons: δ 165.7, 171.2and 175.7 (two peaks are probably overlapping).

(ii) EtOOC—CH₂—CH₂—CH₂—(R)Cha-Pro-Pab×HOAc

EtOOCCH═CHCH₂—(R)Cha-Pro-Pab(Z) (250 mg, 0.38 mmol) was disolved inethanol and hydrogenated in the presence of 5% Pd/C during approximately2 h. Removal of the catalyst by filtration and evaporation of thesolvent in vacuo gave after purification by RPLC using (CH₃CN/0.1 MNH₄OAc) as eluent 70 mg (36%) of the desired product.

¹H NMR (500 MHz, CD₃OD): δ 0.9-1.05 (m, 2H), 1.15-1.55 (m, 5H), 1.25 (t,3H), 1.6-1.85 (m, 7H), 1.95-2.6 (m, 8H), 3.55-3.65 (m, 2H), 3.8 (m, 1H),4.1 (q, 2H), 4.45 (m and d, 2H), 4.55 (d, 1H), 7.55 and 7.75 (two d,4H).

¹³C-NMR (75.0 MHz, CD₃OD): amidine and carbonyl carbons: δ 168.3, 173.2,174.6 and 174.9.

Example 24

Ph(4-COOH)—SO₂—(R)Cha-Pro-Pab×HCl

(i) Ph(4-COOH)—SO₂—(R)Cha-Pro-Pab(Z)

64 mg (0.32 mmol) 4-chlorosulfonyl-benzoic acid was added at ice bathtemperature to a solution of 156 mg (0.29 mmol) H—(R)Cha-Pro-Pab(Z) (SeeExample 15) and 59 mg (0.58 mmol) triethyl amine in 4 ml methylenechloride. The mixture was slowly allowed to reach room temperature andafter 24 hours it was washed with water and dried (Na₂SO₄). Removal ofthe solvent in vacuo and purification of the residue by flashchromatography using ethyl acetate/methanol 9:1 followed by methylenechloride/methanol 3:1 as eluents gave 82 mg (39%) of the product.

(ii) Ph(4-COOH)—SO2—(R)Cha-Pro-Pab×HCl

80 mg (0.11 mmol) Ph(4-COOH)—SO₂—(R)Cha-Pro-Pab(Z) was hydrogenated over5% Pd/C in EtOH. The catalyst was filtered off, the solvent evaporatedand the crude product was purified by RPLC using (CH₃CN/0.1 M NH₄OAc1/4) as eluent and finally converted to the hydrochloride salt by freezedrying from HCl which gave 21 mg (29%) of the product.

¹H-NMR (300 MHz, CD₃OD, mixture of two rotamers): δ 0.45-1.82 (m, 13H),1.90-2.30 (m, 4H), 2.95-4.16 (several m, total 3H), 4.35-4.68 (m, 3H),7.54 (d, 2H), 7.74 (d, 1H), 7.80 (d, 1H), 7.90-8.00 (m, 2H), 8.05-8.22(m, 2H)

¹³C-NMR (75 KHz, CD₃OD): amidine and carbonyl carbons: δ 168.4, 173.4,173.9 and 174.2

MS m/z 584 (M⁺+1)

Example 25

H—(R)Cha-Pic-Pab×2 HCl

(i) Boc-(R)Cha-Pic-Pab(Z)

3.57 g (18.6 mmol) EDC was added at −15° C. to a mixture of 7.11 g (18.6mmol) Boc-(R)Cha-Pic-OH (See preparation of starting materials), 9.07 g(74.2 mmol) DMAP and 5.26 g (18.6 mmol) H-Pab(Z) (See preparation ofstarting materials) in 200 ml DMF. The temperature was allowed to riseto 20° C. over night. The solvent was removed in vacuo and toluene andwater was added. The organic phase was washed with water, 1M KHSO₄, 10%Na₂CO₃ and brine. Drying (MgSO₄) and evaporation of the solvent in vacuogave 13.63 g of a residue which was subjected to flash chromatography onsilica gel using ethyl acetate/toluene 2:1 as eluent to give 9.5 g (79%)of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ 0.7-1.0 (m, 2H), 1.0-2.2 (m, 25H), 2.3-2.5(m, 1H), 2.9-3.1 (m, 1H), 3.8 (d, 1H), 4.3 (dd, 1H), 4.4-4.6 (m, 2H),5.1 (s, 2H), 5.1-5.3 (m, 2H), 7.2-7.3 (m, 5H), 7.35 (d, 2H), 7.4-7.5 (m,1H), 7.75 (d, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 156.8, 164.6,168.2, 170.0 and 173.4.

(ii) H—(R)Cha-Pic-Pab(Z)

Hydrogen chloride was bubbled through a solution of 9.5 g (14.7 mmol)Boc-(R)Cha-Pic-Pab(Z) in 100 ml ethyl acetate at room temperature untilsaturation. After 10 minutes Na₂CO₃ solution (10%) was added and theorganic phase which separated was dried (K₂CO₃) and the solvent wasremoved in vacuo to give the title compound in quantitative yield.

¹H-NMR (500 MHz, CD₃OD): δ 0.85-1.05 (m, 2H), 1.15-1.90 (m, 16H),2.25-2.35 (m, 1H), 3.20-3.30 (m, 1H), 3.80-3.90 (d, 1H), 3.90-4.0 (m,1H), 4.4-4.5 (two d, 2H), 4.7 (br s, 5H) 5.15 (s, 2H), 5.20 (m, 1H),7.25-7.45 (m, 7H), 7.85 (d, 2H).

(iii) H—(R)Cha-Pic-Pab×2 HCl

55 mg (0.1 mmol) H—(R)Cha-Pic-Pab(Z) dissolved in a mixture of 5 mlethanol and 0.45 ml 1M hydrochloric acid was hydrogenated in thepresence of 33 mg 10% Pd/C for 1.5 h. Removal of the catalyst byfiltration and evaporation of the solvent in vacuo gave a residue whichwas subjected to RPLC using 0.1 M NH₄OAc/CH₃CN as eluent. The purifiedpeptide was finally converted to the dihydrochloride salt by dissolutionin hydrochloric acid followed by freeze drying. The yield was 17 mg(35%) of the title compound

¹H-NMR (300 MHz, D₂O, 2 rotamers, 3:1 mixture): δ 1.0-2.0 (m, 18H), 2.33(d, 1H), 3.4-3.5 (m, 1H), 3.8-3.9 (m, 1H), 4.4-4.8 (m, 3H), 5.15.5.25(m, 1H), 7.5-7.7 (m, 2H), 7.8-8.0 (m, 2H).

Resolved signals from the minor rotamer appears at δ: 0.5-0.7 (m) and3.0-3.1 (m)

13C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.3, 171.6 and173.6.

Resolved signals for the minor rotamer appears at δ 170.6 and 172.4.

Example 26

HOOC—CH₂—(R)Cha-Pic-Pab×2 HCl

(i) BnOOC—CH₂—(R)Cha-Pic-Pab(z)

A mixture of 742 mg (1.35 mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25),230 ml (1.45 mmol) benzyl bromoacetate and 558 mg (4 mmol) K₂CO₃ in 4 mlacetonitrile was sonicated at 40° C. for 40 minutes. The solvent wasremoved and the residue was subjected to flash chromatography to give720 mg (77%) of the desired product.

¹H-NMR (500 MHz, CDCl₃); δ 0.8-1.0 (m, 2H), 1.1-1.9 (m, 16H), 2.1-2.4(br s, 1 or 2H), 2.4 (d, 1H), 3.0 (m, 1H), 3.25 (d, 1H), 3.45 (d, 1H),3.55-3.65 (m, 1H), 3.7 (m, 1H), 4.35 (dd, 1H), 4.55 (dd, 1H), 4.80 (twod, 2H), 5.2 (s, 2H), 5.3 (m, 1H), 7.2-7.4 (m, 12H), 7.8 (d, 2H).

¹³C-NMR (125 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 167.9,170.5, 173.4 and 175.5.

(ii) HOOC—CH₂—(R)Cha-Pic-Pab×2 HCl

509 mg (0.73 mmol) BnOOC—CH₂—(R)Cha-Pic-Pab(Z) dissolved in 25 mlethanol was hydrogenated in the presence of 259 mg 10% Pd/C for 4 h.Removal of the catalyst by filtration and evaporation of the solvent invacuo gave a residue which was dissolved in distilled water.Hydrochloric acid was added and the solution was finally freeze dried togive 281 mg (79%) of the title compound.

¹H-NMR (500 MHz, D₂O, mixture of rotamers 4:1): major rotamer: δ 1.0-2.0(m, 18H), 2.25-2.40 (m, 1H), 3.4-3.5 (m, 1H), 3.8-3.95 (m, 3H),4.55-4.65 (two d, 2H), 5.15 (m, 1H), 7.55-7.75 (m, 2H), 7.8-8.0 (m, 2H).

¹³C-NMR (125 MHz, D₂O): amidine and carbonyl carbons: δ 167.3, 169.9,170.3 and 173.5.

Resolved signal for the minor rotamer appears at δ 166.9, 169.2 and172.0

Example 27

HOOC—CH₂-(RorS)CE(COOH)—(R)Cha-Pic-Pab/a X 2 KCl

(i) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pic-Pab(Z)

A mixture of 592 mg (1.1 mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25) and332 mg (1.1 mmol) dibenzyl maleate in 1 ml ethanol was kept at roomtemperature for 1 week. The solvent was removed in vacuo and the residuewas subjected to flash chromatography using methanol/methylene chlorideas eluent to give 275 mg (30%) the diastereomeric mixture.

(ii) HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pic-Pab/a×2 HCl

275 mg BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pic-Pab(Z) dissolved in 20 ml 951ethanol was hydrogenated for 18 hours in the presence of 75 mg 10% Pd/C.The mixture was filtered through hyflo and the solvent was removed invacuo. Addition of water followed by freeze drying gave 166 mg ofHOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Pic-Pab. The two diastereomers wereseparated by RPLC using (CH₃CN/0.1 M NH₄OAc 1/4) as eluent followed byfreeze drying from HCl. This diastereomer eluted first from the column.Yield 9 mg.

¹H-NMR (300 MHz, D₂O, mixture of rotamers): δ 1.0-2.0 (m, 18H), 2.25-2.4(m, 1H), 3.0-3.2 (m, 2H), 3.4 (t, 1H), 3.8 (d, 1H), 4.05 (t, 1H),4.5-4.7 (m, 3H), 5.2 (s, 1H), 7.55 (d, 2H), 7.9 (d, 2H).

Resolved signals from the minor rotamer appears at δ 4.0 (t) and 7.7(d).

Example 28

HOOC—CH₂—(RorS)CH(COOH)—(R)Cha-Pic-Pab/b×2 HCl

The title compound was obtained by using the same procedure as describedin Example 27 on HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Pic-Pat. Thisdiastereomer came out after the first one from the column.

1H—NMR (500 MHz, D2O, mixture of rotamers; δ 1.0-2.0 (m, 18H), 2.25-2.4(m, 1H), 3.0-3.2 (m, 2H), 3.5 (t, 1H), 3.85 (d, 1H), 4.15 (s, 1H),4.5-4.7 (m, 3H), 5.15 (s, 1H), 7.55 (d, 2H), 7.8 (d, 2H).

Resolved signals from the minor rotamer appear at δ 4.35 (s), 7.65 (d)and 7.9 (d).

Example 29

HOOC—CH₂—CH₂—(R)Cha-Pic-Pab×2 KCl

(i) BnOOC—CH₂—CH₂—(R)Cha-Pic-Pab(Z)

A mixture of 851 mg (1.55 mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25) and269 mg (1.71 mmol) benzyl acrylate in 5 ml ethanol was kept at roomtemperature for 40 h. The solvent was removed in vacuo and the residuewas subjected to flash chromatography using methylene chloride/methanolas eluent to give 812 mg (74%) of the product.

¹H-NMR (500 MHz, CDCl₃): δ 0.8-1.0 (m, 2H), 1.1-1.9 (m, 16H), 2.3-2.5(m, 3H), 2.6-2.8 (m, 2H), 3.0 (m, 1H), 3.5 (m, 1H), 3.6-3.7 (m, 1H), 4.3(dd, 1H), 4.6 (dd, 1H), 4.95-5.05 (two d, 2H), 5.2 (s, 2H), 5.3 (m, 1H),6.5-6.9 (br s, 1H), 7.0-7.1 (m, 1H), 7.2-7.5 (m, 12H), 7.75-7.85 (d,2H), 9.3-9.7 (br s, 1H).

(ii) HOOC—CH₂—CH₂—(R)Cha-Pic-Pab×2 HCl

780 mg (1.1 mmol) BnOOC—CH₂—CH₂—(R)Cha-Pic-Pab(Z) dissolved in 25 mlethanol was hydrogenated for 4 h in the presence of 306 mg 15% Pd/C. Thecatalyst was removed by filtration and the solvent was removed in vacuo.The residue was dissolved in hydrochloric acid and the resultingsolution was freeze dried to give 481 mg (78%) of the title compound.

¹H-NMR (500 MHz, D₂O): δ 0.95-1.1 (m, 2H), 1.15-1.9 (m, 16H), 2.2-2.3(m, 1H), 2.7-2.8 (t, 2H), 3.2-3.3 (m, 3H), 3.4-3.5 (m, 1H), 3.75-3.85(m, 1H), 4.4-4.6 (m, 3H), 5.15 (m 1H), 7.5-7.6 (m, 2H), 7.8-7.9 (m, 2H),8.6-8.7 (m,

¹³C-NMR (125 MHz, CD₃OD): amidine and carbonyl carbons: δ 170.6, 175.9,179.5 and 183.5.

Example 30

HOOC—CO—(R)Cha-Pic-Pab×HOAc

(i) EtOOC—CO—(R)Cha-Pic-Pab(Z) 0.12 g ethyloxalyl chloride was added toa mixture of 0.42 g (0.77 mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25) and0.21 g (1.5 mmol) X₂CO₃ in 10 ml CH₃CN at room temperature. After 2hours an additional amount of 0.07 g (0.5 mmol) ethyloxalyl chloride wasadded. The mixture was stirred at room temperature over night. Thesolvent was removed in vacuo. and the residue was dissolved in CH₂Cl₂and washed with water. Evaporation and flash chromatography (toluene:ethyl acetate 1:2 followed by CH₂Cl₂: methanol) gave 0.21 g (42%) of theproduct.(ii) HOOC—CO—(R)Cha-Pic-Pab(Z)

0.21 g (0.32 mmol) EtOOC—CO—(R)Cha-Pic-Pab(Z) was dissolved in 3 ml THFand 0.17 g (4.2 mmol) LiOH dissolved in 3 ml water was added. Themixture was stirred at room temperature over night and then poured ontoethyl acetate/water. The phases were separated and the organic phase wasextracted with a KHCO₃-solution. The aqueous phase was acidified with0.5M HCl (pH 1) and extracted with CH₂Cl₂, dried over Na₂SO₄ andevaporated to give 80 mg of the product.

(iii) HOOC—CO—(R)Cha-Pic-Pab×HOAc

HOOC—CO—(R)Cha-Pic-Pab(Z) was hydrogenated over 5% Pd/C in EtOH. Thecatalyst was filtered off and the solvent evaporated. The residue wassubjected to purification by RPLC to give the title compound.

¹H NMR (500 MHz, DMSO-d₆); δ 0.8-1.0 (m, 2H), 1.1-1.75 (m, 15H),1.86-1.94 (m, 1H), 2.13-2.2 (m, 1H), 3.75-3.81 (m, 1H), 4.32, 4.44 (AB,2H), 4.71-4.77 (m, 1H), 4.98-5.02 (m, 1H), 7.41 (d, 2H), 7.75 (d, 2H),8.1-8.15 (m, 1H), 8.22-8.27 (m, 1H), 9.32 (broad s), 9.90 (broad s). Thesignal of one of the protons (3.25) is partially obscured by the solventsignal.

MS m/z 486 (M⁺+1)

Example 31

HOOC—CH₂—CO—(R)Cha-Pic-Pab

(i) MeOOC—CH₂—CO—(R)Cha-Pic-Pab(Z)

0.39 g (0.72 mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25) and 0.9 g (0.8mmol) monomethylmalonate was dissolved in 40 ml CH₂Cl₂ and 0.16 g (0.8mmol) DCC was added. The solution was stirred in room temperature overnight. The precipitated DCU was removed by filtration and the filtratewas washed with 0.3M KHSO4 and KHCO₃-solution and dried (NaSO₄).Evaporation of the solvent followed by flash chromatography usingtoluen/ethyl acetate (1/3) as eluent gave 0.27 g (58%) of the desiredproduct.

(ii) MeOOC—CH₂—CO—(R)Cha-Pic-Pab

90 mg (0.14 mmol) MeOOC—CH₂—CO—(R)Cha-Pic-Pab(Z) was dissolved in 10 mlethanol and was hydrogenated in presence of 5% Pd/C for 5 hours. Removalof the catalyst by filtration and evaporation of the solvent gave 50 mg(70%) of the title product.

¹H NMR (300 MHz, CD₃OD): δ 0.85-1.1 (m, 2H), 1.1-1.9 (m, 16H), 2.35-2.45(m, 1H), 3.2-3.4 (m, 3H), 3.7 (s, 3H), 3.95-4.05 (m, 1H), 4.4-4.55 (m,3H), 5.15-5.25 (m, 1H), 7.4-7.55 (m, 2H), 7.7-7.85 (m, 2H).

¹³C NMR (75 MHz, CD₃OD): amidine and carbonyl carbons: δ 168.2, 168.7,170.0, 172.4 and 174.6.

MS m/z 514 (M⁺+1)

(iii) HOOC—CH₂—CO—(R)Cha-Pic-Pab

To a solution of 0.14 g (0.27 mmol) of MeOOC—CH₂—CO—(R)Cha-Pic-Pab in 5ml methanol was added 2 ml of 0.5 M NaOH at room temperature. Afterstirring for 5 hours water was added and the methanol was removed invacuo. The aqueous phase was freeze dried. The soluble material wasextracted out from the insoluble inorganic salts with absolute ethanol.The remaining solid after evaporation of the ethanol was suspended inwater and 70 mg (52%) of the title compound was isolated by filtration.

¹H NMR (300 MHz, DMSO-d₆); δ 0.8-1.0 (m, 2H), 1.0-1.9 (m, 16H),2.15-2.30 (m, 1H), 2.58, 2.86 (AB, 2H), 3.8-3.95 (m, 1H), 4.2-4.5 (m,2H), 4.7-4.85 (m, 1H), 4.95-5.05 (m, 1H), 7.40 (d, 2H), 7.77 (d, 2H),8.2-8.3 (m, 1H), 9.3-9.4 (m, 1H), 9.90 (broad s, 3H). The signal of oneof the protons (3.21) is partially obscured by the solvent-signal.

¹³C NMR (75 MHz, DMSO-d₆): amidine and carbonyl carbons: δ 165.8, 168.8,169.9, 172.2 and 172.4.

MS m/z 500 (M⁺+1)

Example 32

MeOOC—CH₂—CO—(R)Cha-Pic-Pab

See Example 31 (ii) above.

Example 33

H₂N—CO—CH₂—(R)Cha-Pic-Pab

(i) H₂N—CO—CH₂—(R)Cha-Pic-Pab(Z)

Attempted alkylation of 455 mg (0.83 mmol) H—(R)Cha-Pic-Pab(Z) (SeeExample 25) with 80 mg (0.86 mmol) chloroacetamide in 3 ml acetonitrilein the presence of 395 mg (2.86 mmol) potassium carbonate by sonicationat 40° C. turned out to be an extremly sluggish reaction. Even theaddition of 230 mg (2.6 mmol) lithium bromide did not seem to improvethe reaction rate. However, addition of lithium iodide andheating/sonication gave small amounts of product, according to TLC.Workup by addition of water, extraction with ethyl acetate/toluene,drying of the organic phase (MgSO₄) and removal of the solvent in vacuogave a residue which was subjected to flash chromatography usingMeOH/CH₂Cl₂ as eluent to give 118 mg (24%) of the desired product.

(ii) H₂N—CO—CH₂—(R)Cha-Pic-Pab×2 HCl

118 mg (0.2 mmol) H₂N—CO—CH₂—(R)Cha-Pic-Pab(Z) dissolved in 10 ml 95%ethanol was hydrogenated in the presence of 143 mg 10% Pd/C for 2 h. Themixture was diluted with distilled water and hydrochloric acid andfiltered through hyflo. Freeze drying gave 26 mg (24%) of the desiredproduct.

¹H-NMR (300 MHz, CD₃OD): δ 0.9-1.1 (m, 2H), 1.1-1.9 (m, 16H), 2.3 (d,1H), 3.4 (t, 1H), 3.6 (AB-system, 2H), 3.8 (d, 2H), 4.35 (t, 1H), 4.5(s, 2H), 5.2 (s, 1H), 7.55 (d, 2H), 7.8 (d, 2H).

Example 34

Boc-(R)Cha-Pic-Pab

10 mg (0.015 mmol) Boc-(R)Cha-Pic-Pab(Z) (See Example 25) dissolved in 5ml ethanol was hydrogenated in the presence of 38 mg 10% Pd/C for 4 h.Removal of the catalyst by filtration and evaporation of the solvent invacuo followed by dissolution of the residue in water and freeze dryingyielded 7.6 mg (95%) of the product.

¹H-NMR (300 MHz, CD₃OD): δ 0.9-1.1 (m, 2H), 1.1-1.9 (m, 16H), 2.4 (d,1H), 3.25 (t, 1H), 4.0 (d, 1H), 4.5 (AB-system, 2H), 4.5-4.6 (m, 1H),5.25 (s, 1H), 7.45 (d, 2H), 7.75 (d, 2H).

Example 35

Ac—(R)Cha-Pic-Pab×HCl

(i) Ac—(R)Cha-Pic-Pab(Z)

Acetyl chloride 0.06 g (0.8 mmol) was added to a mixture of 0.37 q (0.68mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25) and 0.19 g (1.35 mmol) K₂CO₃in 10 ml CH₃CN at room temperature. After stirring for an additional 30minutes at room temperature the solvent was removed in vacuo. Theresidue was dissolved in CH₂Cl₂ and washed with water. Evaporation andflash chromatography using a stepwise gradient of CH₂Cl₂/MeOH (99.9/0.1,99.8/0.2, 99.6/0.4, 99.2/0.8 and 98.4/1.6) gave 0.24 g (60%) of theproduct.

(ii) Ac—(R)Cha-Pic-Pab×HCl

Ac—(R)Cha-Pic-Pab(Z) was hydrogenated over 5% Pd/C at atmosphericpressure. After filtration of the catalyst and evaporation of thesolvent the crude material was subjected to purification by RPLC usingCH₃CN/0.1 M NH₄OAc (35/65) as eluent. Removal of the solvent and excessNH₄OAc followed by freeze drying from 1M HCl gave the title compound.

¹H-NMR (300 MHz, CD₃OD): δ 0.85-1.1 (m, 2H), 1.15-2.0 (m, 19H),2.35-2.47 (m, 1H), 3.2-3.33 (m, 1H), 3.95-4.05 (m, 1H), 4.46,4.57 (ABX,2H), 5.16-5.22 (m, 1H), 7.51 (d, 2H), 7.76 (d, 2H), 8.23 (m, 1H). Thesignal of one of the protons is totally obscured by the solvent-signal.

¹³C-NMR (75 MHz, CD₃OD): amidine and carbonyl carbons: δ 168.3, 172.5,173.8, 175.1

MS m/z 456 (M⁺+1)

Example 36

Me—SO₂—(R)Cha-Pic-Pab×HCl

(i) Me—SO₂—(R)Cha-Pic-Pab(Z)

A solution of 48 mg (0.42 mmol) methanesulfonyl chloride in 0.5 mlmethylene chloride was added at 0° C. to a stirred solution of 209 mg(0.382 mmol) H—(R)Cha-Pic-Pab(Z) (See Example 25) and 0.11 ml (0.763mmol) triethyl amine in 5 ml of methylene chloride. The reaction mixturewas allowed to reach room temperature over night. Washing with waterfollowed by drying (Na₂SO₄) and evaporation of the solvent in vacuo gavea residue which was subjected to flash chromatography using ethylacetate/methanol (95/5) as eluent to give 159 mg (67%) of the product.

(ii) Me—SO₂—(R)Cha-Pic-Pab×HCl

150 mg (0.24 mmol) Me—SO₂—(R)Cha-Pic-Pab(z) dissolved in 5 ml 95%ethanol and 1 ml water was hydrogenated in the presence of 5% Pd/C for 4h. Removal of the catalyst by filtration, addition of 0.2 ml 1Mhydrochloric acid and evaporation of the solvent in vacuo gave a residuewhich was dissolved in 2 ml water and freeze dryed to give 116 mg (86%)of the product.

¹H-NMR (500 MHz, CD₃OD): δ 0.90-1.10 (m, 2H), 1.15-1.85 (m, 15H), 1.90(bd, 1H), 2.30 (bd, 1H), 2.85 (s, 3H), 3.35 (dt, 1H), 3.90 (bd, 1H),4.45 (AB-system, 2H) 4.50-4.55 (m, 1H), 5.13 (dd, 1H), 7.50 (d, 2H),7.75 (d, 2H).

¹³C-NMR (125 MHz D₂O): amidine and carbonyl carbons: δ 166.8, 173.0 and174.6.

Example 37

H—(R)Cha-(R,S)betaPic-Pab×2 HCl

(i) Boc-(R)Cha-(R,S)betaPic-Pab(Z)

EDC was added at −18° C. to a stirred solution of 1.0 g (2.6 mmol)Boc-(R)Cha-(R,S)betaPic-OH (See preparation of starting materials), 1.28g (10.5 mmol) DMAP, 0.74 g (2.6 mmol) H-Pab-(Z) (See preparation ofstarting materials) in 35 ml DMF. The reaction mixture was allowed toreach room temperature over night and the solvent was subsequentlyremoved in vacuo. The residue was dissolved in CH₂Cl₂ and the organiclayer was washed succesively with 0.3M KHSO₄, KHCO₃-solution and brine.Drying (Na₂SO₄) and removal of the solvent gave a residue which wassubjected to flash chromatography using heptane:ethyl acetate with 4%methanol as eluent to yield 0.74 g (44%) of the desired product.

(ii) H—(R)Cha-(R,S)betaPic-Pab(Z)

0.68 g (1.05 mmol) Boc-(R)Cha-(R,S)betaPic-Pab(Z) was dissolved in ethylacetate saturated with HCl(g). The solution was stirred for 1 h at roomtemperature. Water was added and the mixture was made alkaline withK₂CO₃. The water phase was extracted with ethyl acetate. The organicphase was then washed with water and dried (Na₂SO₄). Evaporation gave0.5 q (87%) of the desired product.

(iii) H—(R)Cha-(R,S)betaPic-Pab×2 HCl

65 mg (0.19 mmol) H—(R)Cha-betaPic(R,S)-Pab(Z) was dissolved in 7 mlethanol and hydrogenated in presence of 5% Pd/C for 4 hours. Removal ofthe catalyst by filtration, evaporation of the solvent and freeze dryingfrom 1M HCl and water gave 41 mg (71%) of the product.

¹H NMR (300 MHz, D₂O, 2 diastereomers 4/5, and rotamers); δ 0.8-2.16 (m,), 2.5-2.77 (m, 3H), 3.13-3.43 (m, 3H), 3.68-3.94 (m, 1H), 4.18-4.41 (m,1H), 4.41-4.52 (m, 3H), 7.46-7.57 (m, 2H), 7.72-7.83 (m, 2H).

Example 38

HOOC—CH₂—CH₂—(R)Cha-(R,S)betaPic-Pab×2 HCl

(i) BnOOC—CH₂—CH₂—(R)Cha-(R,S)betaPic-Pab(Z)

0.21 g (0.38 mmol) H—(R)Cha-(R,S)betaPic-Pab(Z) (See Example 37) wasdissolved in 2 ml ethanol. 0.68 g (0.42 mmol) benzyl acrylate was addedand the solution was stirred for 5 days. Evaporation and flashchromatography with CH₂Cl₂/MeOH (95/5) as eluent gave 0.19 g (70%) ofthe desired product.

(ii) HOOC—CH₂—CH₂—(R)Cha-(R,S)betaPic-Pab×2 HCl

170 mg (0.24 mmol) BnOOC—CH₂—CH₂—(R)Cha-(R,S)betaPic-Pab(Z) wasdissolved in 10 ml ethanol and hydrogenated in presence of 5% Pd/C for 4hours. Removal of the catalyst by filtration, evaporation of the solventand freeze drying from 1M HCl and water gave 103 mg (77%) of theproduct.

¹H NMR (300 MHz, D₂O, mixture of 2 diastereomers 4/5 and rotamers); δ0.92-2.03 (m, H), 2.51-2.78 (m, 1H), 3.21-3.52 (m, 1H), 3.88-4.01 (m,1H), 4.07-4.3. (m, 2H), 4.4-4.71 (m, 2H), 7.59 (d, 2H), 7.86 (d, 2H)

¹³C NMR (300.13 MHz, 020, mixture of 2 diastereomers 4/5 and rotamers):amidine and carbonyl carbons: δ 167.0, 168.0, 168.1, 175.9, 176.0,176.3, 176.4 and 178.2.

Example 39

HOOC—CH₂—(R)Cha-Val-Pab×2 HCl

(i) Boc-(R)Cha-Val-Pab(Z)

1.77 g (9.2 mmol) EDC was added at −12° C. to a mixture of 3.41 g (9.2mmol) Boc-(R)Cha-Val-OH(See preparation of starting materials), 2.61 g(9.2 mmol) H-Pab(Z)(See preparation of starting materials), and 4.5 g(36.8 mmol) DMAP in 50 ml DMF. The reaction mixture was allowed to reachroom temperature over night and workup by dilution with water wasfollowed by extraction with toluene, ether and ethyl acetate. Subsequentdrying (MgSO₄) of the combined organic extracts, removal of the solventin vacuo and flash chromatography using CH₂Cl₂/MeOH as eluent gave 2.77g (47%) of the desired product.

(ii) H—(R)Cha-Val-Pab(Z)

Hydrogen chloride was bubbled through a solution of 2.77 g (4.4 mmol)Boc-(R)Cha-Val-Pab(Z) in 75 ml ethyl acetate. After 15 minutes sodiumcarbonate solution was added to pH 10 and the aqueous phase wasextracted with ethyl acetate. Drying (potassium carbonate) and removalof the solvent in vacuo gave 1.8 g (77%) of H—(R)Cha-Val-Pab(Z).

(iii) BnOOC—CH₂—(R)Cha-Val-Pab(Z)

A mixture of 326 mg (0.61 mmol) H—(R)Cha-Val-Pab(Z), 105 ml (0.67 mmol)benzyl bromoacetate, and 252 mg (1.83 mmol) potassium carbonate in 2 mlacetonitrile was sonicated for 2.5 h at 40° C. More acetonitrile wasadded, in order to dissolve the product, and the mixture was filteredand the solvent was removed in vacuo. The residue was purified by flashchromatography using methanol/methylene chloride as eluent. The productwas finally crystallised from ethyl acetate to give 124 mg (30%) ofcolourless crystals.

(iv) HOOC—CH₂—(R)Cha-Val-Pab×2 HCl

124 mg (0.18 mmol) BnOOC—CH₂—(R)Cha-Val-Pab(Z) in 20 ml ethanol washydrogenated for 2 hours in the presence of 25 mg 10% Pd/C. 10 ml of THFwas added and the hydrogenation was continued for another 2 hours at0.50° C. The mixture was filtered through hyflo and the filtercake waswashed with dilute hydrochloric acid. The organic solvents were removedfrom the combined filtrates in vacuo. Freeze drying of the remainingsolution yielded 55 mg (50%) of the desired compound.

¹H-NMR (500 MHz, D₂O); δ 0.75-1.4 (m, 12H), 1.5-1.9 (m, 7H), 2.0-2.15(bs, 1H), 3.45 (AB-system, 2H), 4.1 (m, 2H), 4.5 (m, 2H), 7.5 (s, 2H),7.7 (s, 2H), 8.9 (s, 1H).

Example 40

HOOC—CH₂—CH₂—(R)Cha-Val-Pab×2 HCl

(i) H—(R)Cha-(R,S)Val-Pab(Z)

The title compound was prepared by coupling Boc-(R)Cha-Val-OH withH-Pab(Z), using the pivaloyl coupling as described forBoc-(R)Cha-Pic-OMe (See preparation of starting materials). A totalepimerization of the valine occured to give Boc-(R)Cha-(R,S)Val-Pab(Z).The Boc protecting group was removed in the same way as described forBoc-(R)Cha-Val-Pab(Z) (See Example 39) to give the title compound.

(ii) BnOOC—CH₂—CH₂—(R)Cha-(R,S)Val-Pab(Z)

A solution of 1.007 g (1.9 mmol) H—(R)Cha-(R,S)Val-Pab(Z) and 308 mg(1.9 mmol) benzyl acrylate in 3 ml of ethanol was kept at 40° C. overnight. The solvent was removed in vacuo and the residue was purified byflash chromatography using methanol/methylene chloride (10/90) as eluentto give 1.086 g (82%) of the title compound.

(iii) HOOC—CH₂—CH₂—(R)Cha-Val-Pab×2 HCl

1.086 g (1.6 mmol) BnOOC—CH₂—CH₂—(R)Cha-(R,S)Val-Pab(Z) was hydrogenatedin 25 ml THF and 14 ml 0.5 M hydrochloric acid in the presence of 223 mg10% Pd/C for 2 hours. Removal of the catalyst by filtration throughcelite and removal of the THF in vacuo followed by freeze drying of theremaining aqueous solution gave a residue of which approximately 300 mgwas subjected to HPLC using 25% acetonitrile in 0.1 M Ammonium acetatebuffer as eluent. Two main fractions were isolated, of which the secondfraction contained the title compound. 67 mg of title compound, as thedihydrochloride, was isolated.

¹H-NMR (500 MHz, D₂O); δ 1.0-1.15 (m, 12H), 1.2-1.4 (m, 7H), 1.65-1.9(m, 7H), 2.15-2.25 (m, 1H), 2.85 (t, 2H), 3.15-3.2 (m, 1H), 3.3-3.35 (m,1H), 4.15-4.2 (m, 1H), 4.25 (d, 1H), 4.55-4.65 (AB-system, 2H), 7.65 (d,2H), 7.85 (d, 2H).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 167.0, 169.8,173.96 and 174.04.

Example 41

H—(R)Hoc-Aze-Pab×2 HCl

(i) Boc-(R)Hoc-Aze-Pab(Z)

Prepared in the same way as described for Boc-(R)Cha-Pic-Pab(Z) (SeeExample 25) by replacing Boc-(R)Cha-Pic-OH with Boc-(R)Hoc-Aze-OH (Seepreparation of starting materials). The crude product was subjected toflash chromatography (Toluene/EtOAc 116) to give 0.32 g (0.37%) of thedesired product.

(ii) H—(R)Hoc-Aze-Pab(Z)

Boc-(R)Hoc-Aze-Pab(Z) was treated in the same way as describes forBoc-(R)Cha-Pic-Pab(Z) in Example 25 to to give 0.23 g (88%) of the titlecompound.

(iii) H—(R)Hoc-Aze-Pab×2 HCl

20 mg (0.037 mmol) of H—(R)Hoc-Aze-Pab(Z) was dissolved in 3 ml ethanoland hydrogenated in presence of 5% Pd/C for 4 hours at athmosphericpressure. Removal of the catalyst by filtration, evaporation of thesolvent and freeze drying from 1M HCl gave 11 mg (63%) of the product.

¹H NMR (300.13 MHz, D₂O, mixture of two rotamers 3:1): major rotamer: δ0.9-2.1 (m, 15H), 2.4-2.6 (m, 1H), 2.7-3.0 (m, 1H), 4.1-4.3 (m, 1H),4.35-4.56 (m, 1H), 4.65 (s, 2H), 5.0-5.11 (m, 1H), 7.62 (d, 2H), 7.9 (d,2H). The signal of one of the protons is totally obscured by theH-O-D-signal.

Example 42

HOOC—CH₂—CH₂—(R)Hoc-Aze-Pab×2 TFA

(i) BnOOC—CH₂—CH₂—(R)Hoc-Aze-Pab(Z)

0.067 g (0.41 mmol) benzylacrylate was added to a solution of 0.2 g(0.37 mmol) H—(R)Hoc-Aze-Pab(Z) (See Example 41) in 2 ml ethanol (95%)at room temperature.

The reaction was left at room temperature for 5 days. The solvent wasremoved in vacuo and the residue was purified with flash chromatography(CH₂Cl₂: MeOH, 96/4) to give 0.16 g (62%) of the desired product.

(ii) HOOC—CH₂—CH₂—(R)Hoc-Aze-Pab×2 TFA

160 mg (0.23 mmol) BnOOC—CH₂—CH₂—(R)Hoc-Aze-Pab(Z) was dissolved in 10ml ethanol and subjected to hydrogenation at atmospheric pressure inpresence of 5% Pd on charcoal for 3 hours. Removal of the catalyst byfiltration evaporation of the solvent and freeze drying from water andTFA gave 120 mg (87%) of the product.

¹H NMR (300.13 MHz, D₂O 2 rotamers 3:1); major rotamer: δ 0.9-1.9 (m,13H), 1.94-2.16 (m, 2H), 2.38-2.55 (m, 1H), 2.7-2.97 (m, 3H), 3.2-3.44(m, 2H), 4.16 (m, 1M), 4.35-4.58 (m, 2H), 4.65 (s, 2H), 5.0-5.12 (m,1M), 7.63 (d_(,) 2H), 7.87 (d, 2H)

¹³C NMR (300.13 MHz, D₂O): amidine and carbonyl carbons: δ 167.3, 168.7,172.5 and 176.6.

Example 43

HOOC—CH₂—(R,S)CR(COOH)—(R)Hoc-Pro-Pab×2 HCl

(i) Boc-(R)Hoc-Pro-Pab(Z)

Prepared from Boc-(R)Hoc-Pro-OH (See preparation of starting materials)in the same way as described for Boc-(R)Cha-Pic-Pab(Z) in Example 25.Flash chromatography using ethyl acetate as eluent gave 0.886 g (58%) ofthe title compound.

¹H-NMR. (300 MHz, CDCl₃); δ 0.7-0.95 (m, 2H), 0.95-2.1 (m, 27H (thereof1.2 (s, 9H)), 2.1-2.4 (m, 1H), 3.3-3.5 (m, 1H), 3.65-3.95 (m, 1H),4.0-4.2 (m, 1H), 4.2-4.45 (m, 2H), 4.45-4.6 (d, 1H), 5.15 (apparent bs,2H), 5.2-5.3 (d, 1H), 7.1-7.4 (m, 7H), 7.65 (m, 1H), 7.7-7.8 (d, 2H),9.4 (bs, 1H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 156.3, 164.6,168.1, 171.4 and 172.4.

(ii) H—(R)Hoc-Pro-Pab(Z)

40 ml ethyl acetate saturated with hydrogen chloride was added to 0.82 g(1.266 mmol) Boc-(R)Hoc-Pro-Pab(Z) at 0° C. The temperature was allowedto rise to roomtemperature. The reaction was not completed after 1.5 hand therefore hydrogen chloride was bubbled through the reaction mixtureduring 5 minutes. The solvent was evaporated and ethyl acetate andsaturated sodium carbonate was added and the phases were separated. Theorganic phase was washed with brine and dried (Na₂SO₄) and the solventevaporated in vacuo to give the title compound in almost quantitativeyield.

¹H-NMR (300 MHz, CDCl₃); δ 0.75-0.95 (m, 2H), 0.95-2.4 (m, 17H),3.3-3.55 (m, 2H), 3.55-3.7 (m, 1H), 4.25-4.45 (m, 2H), 4.5-4.6 (m, 1H),5.15 (s, 2H), 7.15-7.35 (m, 5H), 7.35-7.45 (m, 2H), 7.6-7.7 (m, 1H),7.7-7.85 (d, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 167.8,171.4 and 175.3.

(iii) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Hoc-Pro-Pab(Z)

To 0.15 g (0.5 mmol) benzyl acrylate in 1.5 ml EtOH (99%) was added0.273 g (0.498 mmol) H—(R)Hoc-Pro-Pab(Z) and the mixture was stirred atroom temperature for 10 days.

The solvent was removed in vacuo and the residue was subjected to flashchromatography, using ethyl acetate as eluent to give 0.103 g (25%) ofBnOOC—CH₂—(R,S)CH(COOBn)—(R)Hoc-Pro-Pab(Z).

¹H-NMR (300 MHz, CDCl₃); δ 0.75-2.05 (m, 18H), 2.3-2.45 (m, 1H),2.45-2.8 (m, 3H), 3.15-3.45 (m, 3H), 3.5-3.65 (m, 1H), 4.3-4.5 (m, 2H),4.55-4.7 (m, 1H), 4.8 (s, 1H), 4.9-5.1 (m, 3H), 5.2 (s, 2H), 7.1-7.2 (m,1H), 7.2-7.4 (m, 13H), 7.4-7.45 (d, 2H), 7.6-7.8 (m, 3H).

(iv) HOOC—CH₂—(R,S)CH(COOH)—(R)Hoc-Pro-Pab×2 HCl

103 mg (0.122 mmol) BnOOC—CH₂—(R,S)CH(COOBn)—(R)Hoc-Pro-Pab(Z) dissolvedin 4 ml ethanol (99.5%) and 0.3 ml chloroform was hydrogenated in thepresence of 111 mg 5% Pd/C for 2 h. Removal of the catalyst byfiltration and evaporation of the solvent followed by dissolving inwater and freeze drying showed incomplete hydrogenation. Thehydrogenation was continued in the presence of ethanol, 1 N HCl and 5%Pd/C for 5 hours.

Removal of the catalyst by filtration and evaporation of the solventfollowed by dissolving in water and freeze drying gave the titlecompound.

¹H-NMR (500 MHz, CD₃OD, mixture of two diastereomers); δ 0.8-1.0 (m,2H), 1.1-1.4 (m, 6H), 1.6-1.8 (m, 5H), 1.9-2.15 (m, 5H) 2.25-2.35 (m,1H), 2.9-3.2 (m, 2H), 3.5-3.65 (m, 1H), 3.7-3.9 (2m, total 1H), 4.15-4.4(2m, total 1H), 4.4-4.6 (m, 4H), 7.5-7.6 (m, 2H), 7.7-7.85 (m, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 167.9, 168.2,168.3, 172.8, 173.6, 174.3 and 174.4. The signals from the twodiastereomers are partly overlapping.

Example 44

HOOC—CH₂-(R)Hoc-Pic-Pab×2 HCl

(i) Boc-(R)Hoc-Pic-Pab(Z)

Prepared from Boc-(R)Hoc-Pic-OH (See preparation of starting materials)and H-Pab(Z) (See preparation of starting materials) in the same way asdescribed for Boc-(R)Cha-Pic-Pab(Z) (See Example 25). Flashchromatography using ethyl acetate as eluent gave 1.3 g (78%) of thetitle compound.

¹H-NMR (300 MHz, CDCl₃): δ 0.75-0.95 (m, 2H), 0.95-2.0 (m, 31H (thereof1.3 (s, 9H)), 2.4-2.5 (n, 1H), 3.0-3.1 (m, 1H), 3.8 (m, 1H), 4.2-4.45(m, 2H), 4.45-4.55 (m, 2H), 5.15 (apparent bs, 3H), 5.25-5.3 (m, 1H),7.0 (bs, 1H), 7.15-7.5 (m, 7H), 7.7-7.85 (d, 2H), 9.45 (bs, 1H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 156.6, 164.7,168.1, 170.0 and 173.0.

(ii) H—(R)Hoc-Pic-Pab(Z)

100 ml ethyl acetate saturated with hydrogen chloride was added to 1.3 g(1.96 mmol) Boc-(R)Hoc-Pic-Pab(Z) at 0° C. The temperature was allowedto rise to roomtemperature. The solvent was evaporated after 40 minutesand ethyl acetate and saturated sodium carbonate was added and thephases were separated. The organic phase was washed with brine and dried(Na₂SO₄) and the solvent evaporated in vacuo to give 0.85 g (77.5%) ofthe product.

¹H-NMR (300 MHz, CDCl₃): δ 0.75-0.95 (m, 2H), 1.05-2.3 (m, 25H),3.0-3.15 (m, 1H), 3.6-3.75 (m, 2H), 4.25-4.4 (m, 2H), 5.15 (apparent bs,3H), 7.05-7.2 (d, 2H), 7.2-7.35 (m, 4H), 7.35-7.4 (d, 1H), 7.6-7.8 (d,2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 167.9,170.8 and 175.7.

(iii) BnOOC—CH₂—(R)Hoc-Pic-Pab(Z)

0.171 g (0.748 mmol) benzyl bromoacetate was added to a mixture of 0.4 g(0.712 mmol) H—(R)Hoc-Pic-Pab(Z) and 0.217 g (1.57 mmol) K₂CO₃ in 7 mlacetonitrile. The mixture was heated to 60° C. in oilbath for 1 h. Thesolvent was removed and ethyl acetate and water was added. The phaseswere separated and the organic phase was washed with brine and dried(Na₂SO₄). Evaporation in vacuo gave 0.626 g of a residue which wassubjected to flash chromatography using ethyl acetate as eluent, to give2 products. The first compound eluated from the column was(BnOOC—CH₂)₂(R)Hoc-Pic-Pab(Z) (0.28 g) and the second compound eluatedwas the title compund (0.27 g).

BnOOC—CH₂—(R)Hoc-Pic-Pab(Z):

¹H-NMR (300 MHz, CDCl₃): δ 0.7-0.95 (m, 2H), 1.0-1.75 (m, 18H), 2.3-2.5(m, 1 or 2H), 2.9-3.05 (m, 1H), 3.2-3.3 (m, 1H), 3.35-3.5 (m, 2H),3.6-3.7 (m, 1H), 4.35,4.55 (ABX-system, 2H), 4.75 (s, 2H), 5.15(apperent s, 3H), 5.25-5.3 (m, 1H), 7.1-7.45 (m, 12H), 7.7-7.8 (d, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.6, 167.9,170.5, 173.4 and 175.0.

(iv) HOOC—CH₂—(R)Hoc-Pic-Pab×2 HCl

259 mg (0.365 mmol) BnOOC—CH₂—(R)Hoc-Pic-Pab(Z) dissolved in 7.8 mlethanol (99.5%) and 1.2 ml hydrogen chloride (1 N) was hydrogenated inthe presence of 280 mg 5% Pd/C for 4 h. Removal of the catalyst byfiltration and evaporation of the solvent followed by dissolving inwater and freeze drying gave 170 mg (83%) of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ 0.4-1.85 (m, 20H), 1.85-2.2 (m, 1H), 2.9-3.2(m, 1H), 3.4-3.9 (m, 3H), 4.05-4.3 (m, 2H), 4.3-5.05 (m, 2H), 7.1-7.4(m, 2H), 7.4-7.7 (m, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 167.8, 168.6,169.6 and 172.3.

Example 45

(HOOC—CH₂)₂—(R)Hoc-Pic-Pab×2 HCl

(i) (BnOOC—CH₂)₂(R)Hoc-Pic-Pab(Z)

The title compound was obtained in the alkylation of H—(R)Hoc-Pic-Pab(Z)as described in Example 44 above.

¹H-NMR (300 MHz, CDCl₃): δ 0.7-0.95 (m, 2H), 0.95-1.95 (m, 18H),2.35-2.5 (m, 1H), 2.9-3.05 (m, 1H), 3.5-3.85 (m, 6H), 4.35-4.55 (m, 2H),4.9 (2s, 4H), 5.2 (s, 2H), 5.25-5.35 (m, 1H), 7.1-7.45 (m, 16H),7.5-7.65 (m, 1H), 7.7-7.85 (d, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.7, 167.9,170.5, 172.0 and 172.4.

(ii) (HOOC—CH₂)₂—(R)Hoc-Pic-Pab×2 HCl

153 mg (0.178 mmol) (BnOOC—CH₂)₂—(R)Hoc-Pic-Pab(Z) dissolved in 4.5 mlethanol (99.5%) and 0.5 ml hydrogen chloride (1 N) was hydrogenated inthe presence of 150 mg 5% Pd/C for 3.5 h. Removal of the catalyst byfiltration and evaporation of the solvent followed by dissolving inwater and freeze drying gave 109 mg (99%) of (HOOC—CH₂)₂—(R)Hoc-Pic-Pabdihydrochloride. This crude material (80% purity) was subjected toputification by RPLC using CH3CN/0.1 M NH4OAc, 1:4 as eluent. Removal ofthe solvent and excess NH4OAc followed by freeze drying from 1 M HClgave the title compound.

¹H-NMR (500 MHz, D₂O, mixture of two rotamers): major rotamer: &0.95-2.15 (m, 20H), 2.25-2.35 (m, 1H), 3.45-3.55 (m, 1H), 3.95-4.25 (m,5H), 4.6-4.65 (m, 2H), 4.92-5.01 (m, 1H) 5.15-5.20 (m, 1H), 7.58-7.63(d, 2H), 7.84-7.89 (d, 2H).

Resolved signals arising from the minor rotamer appears at: δ 0.7-0.85(m), 2.35-3.45 (m), 3.05-3.15 (m), 4.47-4.55 (m), 4.55-4.6 (m), 4.65-4.7(m), 7.63-7.67 (d), 7.89-7.95 (d).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 168.20, 169.70,170.20 and 172.71.

Example 46

HOOC—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab×2 HCl

(i) Boc-(R)Pro(3-(S)Ph)-Pro-Pab(Z)

To a solution of 570 mg (1.5 mmol) Boc-(R)Pro(3-(S)Ph)-Pro-OH (Seepreparation of starting materials), 425 mg (1.5 mmol) H-Pab(Z) (Seepreparation of starting materials) and 733 mg (6 mmol) DMAP in 25 mlCH₃CN/DMF (1.5/1) was added 310 mg (1.62 mmol) EDC and the mixture wasstirred for 23 h at room temperature. Most of the solvent was evaporatedand 50 ml water was added to the residue. The water phase was extractedwith 1×75 and 2×50 ml EtOAc. The combined organic phase was washed with1×20+1×10 ml 1M KHSO₄, 1×15 ml NaHCO₃(aq), 3×15 ml water, 1×15 ml brineand dried (MgSO₄). Filtration and evaporation of the solvent gave 670 mgof an oil which was purified by flash chromatography using EtOAc aseluent which gave 529 mg (55%) of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ 1.26 (s, 9H), 1.53-1.88. (m, 3H), 2.1-2.31(m, 3H), 2.52 (q, 1H), 3.58-3.77 (m, 4H), 4.31 (d, 1H), 4.35 and 4.47(ABX-system, 2H), 4.65 (dd, 1H), 5.19 (s, 2H), 7.1-7.37 (m, 10H), 7.42(d, 2H), 7.81 (d, 2H), 8.0 (t, 1H(NH)).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 154.6, 164.6,168.1, 171.1 and 171.3.

(ii) H—(R)Pro(3-(S)Ph)-Pro-Pab(Z)

529 mg (0.81 mmol) of Boc-(R)Pro(3-(S)Ph)-Pro-Pab(Z) was dissolved in 15ml EtOAc/HCl(g,saturated) at room temperature and stirred for 3 h. Thesolvent was evaporated and the residue was dissolved in 70 ml CH₂Cl₂.the organic phase was washed with 1×10 ml 2 M NaOH, 1×10 ml water, 1×10ml brine and dried (MgSO₄). Filtration and evaporation of the solventgave 403 mg (90%) of the title compound as a white powder.

¹H-NMR (300 MHz, CDCl₃): δ 1.44-1.57 (m, 1H), 1.62-1.86 (m, 2H),1.96-2.35 (m, 3H), 2.45 (q, 1H), 3.05-3.35 (m, 4H), 3.83 (bd, 1H),4.25-4.45 (m, 2H), 4.53 (m, 1H), 5.19 (s, 2H), 7.16-7.37 (m, 10H), 7.42(d, 2H), 7.66 (t, 1H,(NH)), 7.77 (d, 2H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.4, 167.9,171.1 and 173.0.

(iii) BnOOC—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab(Z)

A mixture of 200 mg (0.36 mmol) H—(R)Pro(3-(S)Ph)-Pro-Pab(Z), 105 mg(0.46 mmol) Br—CH₂—COOBn and 125 mg (0.90 mmol) K₂CO₃ in 10 ml CH₃CN washeated to 50° C. for 1 h and 30 minutes. The solvent was evaporated andthe residue was dissolved in 70 ml EtOAc. The organic phase was washedwith 10 ml water and dried (MgSO₄). Filtration and evaporation of thesolvent gave 260 mg of an oil. The crude material was purified by flashchromatography using a stepwise gradient of CH₂Cl₂/MeOH(NH₃-saturated)(95/5 followed by 9/1) to give 182 mg (72%) of the title compound as awhite solid.

¹H-NMR (300 MHz, CDCl₃): δ 1.43-1.82 (m, 3H), 1.96-2.13 (m, 1H),2.14-2.22 (m, 1H), 2.26-2.43 (m, 2H), 3.02-3.14 (m, 2H), 3.24-3.51 (m,4H), 3.83 (d, 1H), 4.29-4.46 (ABX-system centered at 4.37, 2H), 4.58(dd, 1H), 4.97-5.1 (AB-system centered at 5.03, 2H), 5.19 (s, 2H),7.16-7.38 (m, 15H), 7.43 (d, 2H), 7.5-7.8 (m, 3H, one NH).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 167.9,171.15, 171.2 and 172.7.

(iv) HOOC—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab×2 HCl

0.18 g (0.26 mmole) of BnOOC—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab(Z) was mixedwith 0.075 g 5% Pd/C, 1.0 ml 1N HCl-solution, 1 ml water and 10 mlethanol and the mixture was hydrogenated at atmospheric pressure for onehour. Filtration of the catlyst through hyflo, evaporation of thesolvent followed by freeze drying twice from water gave 129 mg of acrude product. The crude product was purified by RPLC using a stepwisegradient of 0.1 M NH₄OAc/CH₃CN 4/1 followed by 3/1. Evaporation followedby freeze drying from water and 1N HCl-solution gave 70 mg (50%) of thepure product.

¹H-NMR (300 MHz, D₂O): δ 1.42-1.60 (m, 1H), 1.65-1.83 (m, 1H), 1.83-1.98(m, 1H), 2.03-2.20 (m, 2H), 2.63 (t, 2H), 3.28-3.40 (m, 1H), 3.55-3.78(m, 2H), 3.81-3.96 (AB-system central at δ 3.88, 2H), 4.06-4.19 (m, 1H),4.37-4.61 (AB-system central at δ 4.49, 2H), 4.48 (dd, 1H), 4.70 (d,1H), 7.35-7.58 (m, 7H), 7.74 (d, 2H)

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 167.02, 167.2,169.3 and 174.4.

Example 47

HOOC—CH₂—CH₂—(R)Pro(3-(S)-Pro-Pab×2 HCl

(i) BnOOC—CH₂—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab(Z)

To a solution of 190 mg (0.34 mmol) H—(R)Pro(3-(S)Ph)-Pro-Pab(Z) (SeeExample 46) in 7 ml EtOH (99%) was added 114 mg (0.70 mmol) of benzylacrylate and the reaction mixture was stirred at room temperature for 24h. Evaporation of the solvent followed by flash chromatography using astepwise gradient of CH₂Cl₂/MeOH(NH₃-saturated) (95/5 followed by 9/1)gave 202 mg (83%) of the title compound.

¹H-NMR (300 MHz, CDCl₃): δ 1.5-1.71 (m, 2H), 1.74-1.9 (m, 1H), 1.9-2.05(m, 1H), 2.2-2.64 (m, 5H), 2.69-2.82 (m, 2H), 2.84-2.96 (m, 1H),3.18-3.48 (m, 4H), 4.28-4.44 (m, 2H), 4.61 (m, 1H), 4.48-5.08 (AB-systemcentered at 5.03, 2H), 5.19 (s, 2H), 7.15-7.37 (m, 15H), 7.44 (d, 2H),7.75-7.85 (m, 3H, one NH).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.6, 168.0,171.2, 172.5 and 172.9.

(ii) HOOC—CH₂—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab×2 HCl

0.20 g (0.28 mmole) of BnOOC—CH₂—CH₂—(R)Pro(3-(S)Ph)-Pro-Pab(Z) wasmixed with 0.075 g 5% Pd/C, 1.0 ml 1N HCl-solution, 1 ml water and 10 mlethanol and the mixture was hydrogenated at atmospheric pressure for onehour. Filtration of the catalyst through hyflo, evaporation of thesolvent followed by freeze drying twice from water gave 125 mg 79% ofthe title compound.

H-NMR (300 MHz, D₂O): δ 1.44 (m, 1H), 1.65-1.9 (m, 2H), 2.0-2.2 (m, 2H),2.62 (q, 2H), 2.83 (t, 2H), 3.27-3.4 (m, 1H), 3.4-3.8 (m, 4H), 4.0-4.15(m, 1H), 4.35-4.6 (m, 3H), 4.68 (d, 1H), 7.35-7.6 (m, 7H), 7.77 (d, 2H)

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 166.2, 167.1,174.1 and 174.2.

Example 48

HOOC—CH₂—CH₂—(R)Tic-Pro-Pab×2 HCl

(i) Boc-(R)Tic-Pro-Pab(Z)

Prepared in the same way as described for Boc-(R)Cha-Pic-Pab(Z) (SeeExample 25) using Boc-(R)Tic-Pro-OH(See preparation of startingmaterials) instead of Boc-(R)Cha-Pic-OH. Flash chromatography usingheptane/EtOAc (4/1) followed by EtOAc as eluents gave 425 mg (37%) ofthe title compound.

¹H NMR (500 MHz, CDCl₃): δ 1.35 (s, 9H), 1.95-2.15 (m, 3H), 2.4 (m, 1H),2.8 (m, 1H), 3.3 (m, 1H), 3.55 (m, 2H), 4.25-4.4 (two m, 2H)₁ 4.55-4.7(two m, 2H), 7.15-7.5 (m, 10H), 7.85 (d,2H).

¹³C-NMR (75.0 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.6, 171.5and 171.6. (two peaks are probably overlapping)

(ii) H—(R)Tic-Pro-Pab(Z)

Boc-(R)Tic-Pro-Pab(Z) (379 mg, 0.59 mmol) was dissolved in EtOAcsaturated with HCl(g) and stirred at room temperature. Evaporation ofthe solvent gave 251 mg (79%) of the title compound as a white powder.

¹H NMR (500 MHz, CDCl₃): δ 1.65-2.15 (two m, 7H), 2.45 (m, 1H), 2.75 (n,1H), 2.9 (m, 1H), 3.0 (m, 1H), 3.25 (m, 1H), 3.55 (m, 1H), 3.85 (m, 1H),4.35-4.55 (m, 2H), 4.75 (d, 1H), 4.9 (s, 1H), 5.25 (s, 2H), 6.8-7.45(several m, 8H), 7.5 and 7.85 (two d, 4H).

¹³C-NMR (75.0 MHz, CDCl₃): amidine and carbonyl carbons: δ 164.5, 171.3and 172.7 (two peaks are probably overlapping).

(iii) BnO₂C—CH₂—CH₂—(R)Tic-Pro-Pab(Z)

H—(R)Tic-Pro-Pab(Z) (140 mg, 0.26 mmol) was treated with benzyl acrylat(63 mg, 0.39 mmol) in EtOH (1.3 ml) at 20° C. during 48 h. Evaporationof the solvent and flash chromatography using (50% EtOAc/Heptan then 10%MeOH/EtOAc) as eluent afforded 133 mg (73%) of the desired product as awhite solid material.

¹H NMR (500 MHz, CDCl₃): δ 1.75-2.0 (two m, 4H), 2.25 (m, 1H), 1.4-1.65(m, 3H), 2.7-2.95 (two m, 4H), 3.05-3.2 (n, 2H), 3.9 (m, 1H), 4.45 (m,2H), 4.65 (m, 1H), 5.1 (two d, 2H), 5.25 (s, 2H), 6.85-7.45 (several m,12H), 7.5 and 7.9 (two d, 4H).

¹³C-NMR (75.0 MHz, CDCl₃): amidine and carbonyl carbons: δ 171.5, 171.9and 172.1 (two peaks are probably overlapping).

(iv) HOOC—CH₂—CH₂—(R)Tic-Pro-Pab×2 HCl

BnO₂C—CH₂—CH₂—(R)Tic-Pro-Pab(Z) (125 mg, 0,17 mmol) was hyrogenated over5% Pd/C using EtOH/HCl as solvent. Filtration of the catlyst and freezedrying gave 73 mg (77%) of the title compound as a white powder.

¹H NMR (500 MHz, D₂O): δ 2.1-2.35 (two m, 3H), 2.6 (m, 1H), 2.95-3.1 (m,2H), 3.25-3.5 (two m, 2H), 3.65 (m, 3H), 4.65 (s, 2H), 4.75 (m, 1H),5.85 (s, 1H), 7.15-7.6 (three m, 4H), 7.6 and 7.85 (two d, 4H).

¹³C-NMR (75.0 MHz, D₂O): amidine and carbonyl carbons: δ 166.9, 167.1and 174.3 (two peaks are probably overlapping).

Example 49

HOOC—CH₂—CH₂—(R)Cgl-Aze-Pig×2 HCl

(i) Boc-(R)Cgl-Aze-Pig(z)₂

To a mixture of 0.623 g (1.83 mmole) Boc-(R)Cgl-Aze-OH(See preparationof starting materials), 0.816 g (1.92 mmole) H-Pig(Z)₂ (See preparationof starting materials) and 0.89 g (7.3 mmole) DMAP in 10 mldichloromethane was added 0.368 g (1.92 mmole) of EDC and the mixturewas stirred over night. The mixture was diluted and washed with 0.3 MKHSO₄ and once with brine. The organic layer was dried (Na₂SO₄),filtered and evaporated to yield 1.4 g of a crude product. Purificationby flash chromatography using ethyl acetate as eluent gave 0.3 g (22%)of the pure product.

(ii) H—(R)Cgl-Aze-Pig(z)₂

0.3 g (0.4 mmole) Boc-(R)Cgl-Aze-Pig(Z)₂ was mixed with 10 mldichloromethane and 2.5 ml trifluoroacetic acid. The mixture was stirredfor one and a half hour. After evaporation of the solvent the residuewas dissolved in dichloromethane and washed twice with 0.2 MNaOH-solution. The combined water layer was extracted one more time withdichloromethane. The combined organic layer was dried (Na₂SO₄), filteredand evaporated to yield 0.24 g (93%) of the product.

¹H-NMR (300 MHz, CDCl₃, 339K): δ 0.9-1.9 (m, 15H), 1.94 (bd, 1N),2.37-2.52 (m, 1H), 2.65-2.8 (m, 1H), 2.9-3.08 (m, 3H), 3.20 (t, 2H),1.05-4.28 (m, 4H), 4.86 (dd, 1H), 5.16 (s, 4H), 7.2-7.42 (m, 10H), 7.98(bs, NH).

(iii) BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pig(Z)₂

0.231 g (0.36 mmole) was dissolved in 2 ml ethanol and 61 μl (0.40mmole) bensylacrylate was added. The reaction mixture was stirred forfive days at room temperature. The mixture was evaporated and the crudeproduct purified by flash chromatography using a stepwise gradient ofCH₂Cl₂/MeOH (95/5, 90/10) as eluent to yield 0.218 g (75%) of the pureproduct.

¹H-NMR (300 MHz, CDCl₃, 335K): 0.93 (bq, 1H), 1.02-1.85 (m, 14H), 1.94(bd, 1H), 2.33-2.5 Cm, 3H), 2.58-2.77 (m, 2H), 2.79-3.02 (m, 4H), 3.17(t, 2H), 4.0-4.25 (m, 4H), 4.86 (dd, 1H), 5.11 (s, 2H), 5.12 (s, 4H),7.2-7.4 (m, 15H), 8.03 (bs, NH), 10.35 (bs, NH)

(iv) HOOC—CH₂—CH₂—(R)Cgl-Aze-Pig×2 HCl

0.218 g (0.27 mmole) of BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pig(Z)₂ was mixed with0.10 g 5% Pd/C, 1 ml 1M HCl-solution, 1 ml water and 10 ml ethanol andthe mixture was hydrogenated at atmospheric pressure for one hour.Filtration of the catalyst through hyflo, evaporation of the solventfollowed by freeze drying twice from water gave 134 mg (95%) of thetitle compound.

¹H-NMR (300 MHz, D₂O): δ 1.0-1.4 (m, 7H), 1.55-2.05 (m, 9H), 2.22-2.34(m, 1H), 2.61-2.76 (m, 1H), 2.88 (t, 2H), 3.08 (bt, 2H), 3.19 (d, 2H),3.34 (m, 2H), 3.83 (bd, 2H), 3.95 (d, 1H), 4.29-4.49 (m, 2H), 4.90 (dd,1H)

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons: δ 156.4, 167.6,172.1 and 174.7

Example 50

HOOC—CH₂—(R)Cgl-Pro-Pig×2 HCl

(i) Boc-(R)Cgl-Pro-Pig(Z)₂

0.568 g (2.96 mmol) EDC was added at −15° C. to a mixture of 1 g (2.82mmol) Boc-(R)Cgl-Pro-OH (See preparation of starting materials), 1.197 g(2.82 mmol) H-Pig(Z)₂ (See preparation of starting materials) and 1.38 g(11.28 mmol) DMAP in acetonitrile. The temperature was allowed to riseto roomtemperature over night. The solvent was evaporated in vacuo andmethylenchloride and 1 M KHSO₄ was added. The phases were separated andthe organic phase was washed with saturated NaHCO₃, water and brine,drying (Na₂SO₄) and evaporation of the solvents gave 2.033 g of aresidue wich was subjected to flash chromatography using ethylacetate aseluent. This gave two products; 720 mg (34%) of the title compound whicheluted first from the column followed by 775 mg (44%) ofBoc-(R)Cgl-Pro-Pig(Z) formed by loss of one of the Z-protecting groups.

¹H-NMR (300 MHz, CDCl₃); Some signals, especially in the piperidin ring,are selectively broader due to an intramolecular exchange process. Thisis especially pronounced for the 2- and 6-CH₂ groups of the piperidinring, wich exhibit a broad peak ranging from 3.5 to 4.5 ppm.

δ 0.85-2.1 (m, 19H), 2.3-2.45 (m, 1H), 2.8-3.2 (m, 4H), 3.45-3.55 (m,1H), 3.55-3.65 (m, minor rotamer), 3.8-3.93 (m, 1H), 3.97-4.1 (m, 1H),4.52-4.62 (d, 1H), 5.1 (apparent bs, 5H), 7.12-7.41 (m, 10H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons: δ 155.2, 156.3,171.0 and 172.1.

(ii) H—(R)Cgl-Pro-Pig(Z)₂

720 mg (0.946 mmol) of Boc-(R)Cgl-Pro-Pig(Z)₂ was dissolved in 35 ml ofTFA/CH₂Cl₂, 1/4 and stirred for 30 minutes. The solvent was removed invacuo and ethylacetate and 2M NaOH was added. The organic layer waswashed with water and brine, dried (Na₂SO₄) and the solvent wasevaporated in vacuo to give the title compound in quantitative yield.

¹H-NMR (300 MHz, CDCl₃); Some signals, especially in the piperidin ring,are selectively broader due to an intramolecular exchange process. Thisis especially pronounced for the 2- and 6-CH₂ groups of the piperidinring, wich exhibit a broad peak ranging from 3.5 to 4.5 ppm.

δ 0.8-2.15 (m, 19H), 2.22-2.4 (m, 1H), 2.75-2.98 (m, 2H), 2.98-3.18 (m,2H), 3.18-3.35 (m, 1H), 3.35-3.5 (qvart., 1H), 3.5-3.7 (m, 1H),4.42-4.58 (d, 1H), 5.1 (s, 4H), 7.1-7.5 (m, 10H).

¹³C-NMR (75 MHz, CDCl₃): amidine and carbonyl carbons; δ 154.96, 171.31,174.82.

(iii) BnOOC—CH₂—(R)Cgl-Pro-Pig(Z)₂

0.298 g (0.999 mmol) BnOOC—CH₂—OTf (see preparation of startingmaterialswas added to a mixture of 0.64 g (0.999 mmol) H—(R)Cgl-Pro-Pig(Z)₂ and0.531 g (2.996 mmol) K₂CO₃ in 6.4 ml acetonitrile and heated to reflux.After 1 h 20 min the mixture was washed with water, dried (Na₂SO₄) andthe solvent evaporated in vacuo to give 729 mg of a residue which wassubjected to flash chromatography using ethylacetate as eluent. Thisgave two products: 120 mg of (BnOOC—CH₂)₂—(R)Cgl-Pro-Pig(Z)₂ whicheluted first from the column and 142 mg (18%) of the title compound.

¹H-NMR (300 MHz, CDCl₃); Some signals, especially in the piperidin ring,are selectively broader due to an intramolecular exchange process. Thisis especially pronounced for the 2- and 6-CH₂ groups of the piperidinring, wich exhibit a broad peak ranging from 3.5 to 4.6 ppm.

δ 0.94-2.27 (m, 19H), 2.28-2.43 (m, 1H), 2.8-2.98 (m, 2H), 2.98-3.06 (m,1H), 3.06-3.15 (d, 1H), 3.15-3.25 (m, 1H), 3.3-3.5 (m, 4H), 4.5-4.61 (d,1H), 5.1 (s, 6H), 7.1-7.6 (m, 15H), 10.52 (bs, 1H).

(iv) HOOC—CH₂—(R)Cgl-Pro-Pig×2 HCl

142 mg (0.176 mmol) BnOOC—CH₂—(R)Cgl-Pro-Pig(Z)₂ was hydrogenated in thepresence of 0.88 ml 1 M hydrochloric acid, 10 ml ethanol (99.5%) and 180mg 5% Pd/C for 2 h. Removal of the catalyst by filtration on hyflo andmillipore filter followed by evaporation of the solvent in vacuo andfreeze drying gave 95 mg of HOOC—CH₂—(R)Cgl-Pro-Pig×2 HCl. This crudematerial (79% purity) was purified on RPLC using CH₃CN/0.1H NH₄OAc 15/85as eluent. Removal of the solvent and excess NH₄OAc by freeze drying,conversion to hydrochloric acid salt by dissolving in 1 M hydrochloricacid followed by freeze drying gave the title compound.

¹H-NMR (500 MHz, D₂O); δ 1.1-1.35 (m, 6H), 1.63-2.14 (m 13H), 2.26-2.36(m, 1H), 3.01-3.23 (m, 4H), 3.49-3.62 (qvart., 2H), 3.62-3.77 (m, 2H),3.77-3.88 (apparent d, 2H), 4.18-4.32 (d, 1H), 4.37-4.5 (m, 1H).

Example 51

H—(R)Cha-Aze-Pig×2 HCl

(i) Boc-(R)Cha-Aze-Pig(Z)₂

To a well stirred mixture of 86 mg (0.243 mmol) Boc-(R)Cha-Aze-OH (Seepreparation of starting materials), 100 mg (0.236 mmol) H-Pig(Z)₂ (Seepreparation of starting materials) and 115 mg (0.944 mmol) DMAP in 5 mlCH₃CN was added 50 mg (0.260 mmol) EDC and the reaction was stirred for20 h at room temperature. The solvent was evaporated and the residue wasdissolved in 70 ml EtOAc and the organic phase was washed with 3×5 ml 1M KHSO₄, 1×5 ml NaHCO₃, 3×5 ml H₂O, 1×5 ml brine and dried (MgSO₄).Filtration and evaporation of the solvent gave 141 mg of an oil. Thecrude product was purified by flash chromatography (36 g SiO₂) using astepwise gradient of CH₂Cl₂/MeOH (97/3 followed by 95/5) to yield 43 mg(24%) of the title compound.

(ii) H—(R)Cha-Aze-Pig(z)₂

Hydrogen chloride was bubbled through a mixture of 43 mg (0.0565 mmol)Boc-(R)Cha-Aze-Pig(Z)₂ in 10 ml of ethylacetate during 5 minutes. Thesolvent was evaporated in vacuo and ethyl acetate and 0.1 MNaOH-solution was added. The phases were separated and the organic phasewas washed with water and brine and dried (Na₂SO₄). The solvent wasevaporated to give 38 mg, wich was subjected to flash chromatographyusing 10% NH3-saturated methanol in ethyl acetate as eluent to give 28mg of the desired product.

¹H-NMR (300 MHz, CDCl₃); Some signals, especially in the piperidin ring,are selectively broader due to an intramolecular exchange process. Thisis especially pronounced for the 2- and 6-CH₂ groups of the piperidinring, wich exhibit a broad peak ranging from 3.7 to 4.5 ppm.

δ 0.75-1.85 (m, 18H), 2.35-2.53 (m, 1H), 2.62-2.78 (m, 1H), 2.8-3.0 (m,2H), 3.0-3.28 (m, 2H), 3.28-3.3.7 (m, 1H), 3.97-4.18 (m, 2H), 4.8-4.9(m, 1H), 5.1 (s, 4H), 7.2-7.45 (m, 9H), 8.05-8.15 (m, 1H).

(iii) H—(R)Cha-Aze-Pig×2 HCl

28 mg (0.042 mmol) H—(R)Cha-Aze-Pig(Z)₂ dissolved in 2 ml ethanol(99.5%) and 0.13 ml hydrogen chloride (1 N) was hydrogenated in thepresence of 35 mg 5% Pd/C for 4 h. Removal of the catalyst by filtrationand evaporation in vacuo of the solvent followed by dissolving in waterand freeze drying gave 12 mg (60. %) of H—(R)Cha-Aze-Pigdihydrochloride.

¹H-NMR (500 MHz, 300 K, CD₃OD); Some signals, especially in thepiperidin ring, are selectively broader due to an intramolecularexchange process. This is especially pronounced for the 2- and 6-CH₂groups of the piperidin ring, wich exhibit a broad peak ranging from 3.7to 4.5 ppm.

δ 0.75-2.1 (m, 18H), 2.2-2.35 (m, 1H), 2.62-2.75 (in, 1H), 3.0-3.12 (t,2H), 3.12-3.23 (d, 2H), 3.85-3.95 (d, 2H), 3.95-4.0 (dd, 1H), 4.15-4.23(m, 1H), 4.35-4.42 (m, 1H), 4.72-4.78 (m, 1H).

¹³C-NMR (75 MHz, CD₃OD): guanidine: δ 157.6; carbonyl carbons: δ 170.0and 172.6.

Example 52

HOOC—CH₂—(R)Cgl-Aze-Pac×2 HCl

(i) Boc-(R)Cgl-Aze-Pac(Z)

To a solution of 0.47 g (1.4 mmol) of Boc-(R)Cgl-Aze-OH (See preparationof starting materials), 0.40 g (1.4 mol) of H-Pac(Z)(See preparation ofstarting materials) and 0.67 g (5.5 mmol) of DMAP in 5 ml of acetonitrilwas added 0.27 g of EDC at 0° C. The mixture was stirred at roomtemperature over night and subsequently diluted with ethyl acetate. Thesolution was washed with KHSO₄ (aq) and NaHCO₃ (aq), dried (Na₂SO₄),filtered and evaporated. Flash chromatography using ethyl acetatefollowed by ethyl acetate/methanol 98/2 as eluents gave 0.25 g (301) ofthe title compound as a mixture of 1,4-cis- and trans-products withrespect to the Pac part of the molecule.

¹H-NMR (500 MHz, CDCl₃): δ 0.8-2.0 (m, 29H; thereof 1.45 (s, 9H)), 2.15and 2.34 (m, 1H, isomers), 2.45-2.7 (m, 2H), 3.0-3.4 (m, 2H), 3.85 (m,1H), 4.14 (m, 1H), 4.33 (m, 1H), 4.85 (m, 1H), 4.98 (m, 1H), 5.04 (s,2H), 7.25-7.45 (m, 5H), 7.8-7.9 (m, 1H), 9.2-9.5 (m, 1H).

(ii) H—(R)Cgl-Aze-Pac(Z)×HCl

Boc-(R)Cgl-Aze-Pac(Z), 0.25 g (0.41 mmol), was dissolved in 100 ml ofethyl acetate and cooled in an ice bath. HCl (g) was bubbled through for5 min and the solvent was evaporated.

¹H-NMR (300 MHz, MeOD): δ 0.8-2.0 (m, 22H), 2.05-2.35 (m, 1H), 2.4-2.55(m, 1H), 2.6-2.75 (M, 1H), 3.00 (d, 1H), 3.05 and 3.37 (multiplets, 0.6Hand 0.4H respectively, isomers), 3.15-3.3 (m, 1H), 4.05-4.2 (m, 2H),4.88 (dd, 1H), 5.11 (s, 2H), 7.2-7.45 (m, 5H), 8.0-8.15 (m, 1H).

(iii) BnO₂C—CH₂—(R)Cgl-Aze-Pac(Z)

A mixture of 0.17 g (0.33 mmol) of H—(R)Cgl-Aze-Pac(Z)×HCl, 0.11 g (0.37mmol) of benzyl triflyloxyacetate and 0.14 g (1.0 mmol) of K₂CO₃ in 5 mlof acetonitrile was stirred at room temperature for 3 days. The crudematerial was flash chromatographed with EtOAc/CH₂Cl₂/MeOH 95/20/5.Yield: 70 mg (32%)

¹H-NMR (500 MHz, CDCl₃): δ 0.85-2.3 (m, 20H), 2.48 (m, 1H), 2.63 (m,1H), 2.87 (m, 1H), 3.05-3.25 (m, 1H), 3.25-3.35 (m, 2H), 3.38 (dd, 1H),3.95 (m, 1H), 4.08 (m, 1H), 4.88 (m, 1H), 5.1-5.2 (m, 4H), 5.9-6.3 (m,1H), 7.25-7.5 (m, 10H), 8.00 and 8.08 (broad triplets, 1H, isomers).

(iv) HO₂C—CH₂—(R)Cgl-Aze-Pac×2 HCl

BnO₂C—CH₂—(R)Cgl-Aze-Pac(Z), 70 mg (0.11 mmol), was dissolved in 5 ml ofethanol, and 5% Pd/C and 0.1 ml of conc. HCl were added. The mixture washydrogenated at atmospheric pressure for 1 h. After filtration andevaporation the product was purified through preparative RPLC using 0.1M NH₄OAc/CH₃CN 4/1 as eluent. After change of salt to the hydrochlorideand freeze drying the title compound was obtained as a 45/55 mixture of1,4-cis- and trans-isomers with respect to the Pac part of the molecule.Yield: 40 mg (74%).

¹H-NMR (500 MHz, D₂O) δ 1.1-2.1 (m, 20H), 2.32 (m, 1H), 2.52 (m, 1H),2.63 (m, 1H), 2.72 (m, 1H), 3.1-3.3 (m, 1H), 3.40 (m, 1H), 3.8-3.95 (m,2H), 4.04 (d, 1H), 4.39 (m, 1H), 4.93 (m, 1H).

¹³C-NMR (125 MHz, D₂O) amidine and carbonyl carbons: δ 167.7, 172.0,174.9 and 175.2.

Example 53

H—(R)Cha-Pro-Pac×2 HCl

(i) Boc-(R)Cha-Pro-Pac(Z)

211 mg (1.1 μmmol) EDC was added at 0° C. to a stirred solution of 0.4 g(1.1 mmol) H-Pac(Z)×2 HCl (See preparation of starting materials), 0.4 g(1.1 mmol) Boc-(R)Cha-Pro-OH(See preparation of starting materials), and0.55 g DMAP in 7 ml acetonitrile. The reaction mixture was stirred at 0°C. for 1 h and at room temperature for 2 h. The solvent was removed invacuo and the residue was diluted with ethyl acetate and water. Theorganic phase was washed with acetic acid, water, and sodium hydrogencarbonate solution and dried (MgSO₄). Removal of the solvent in vacuogave a residue which was purified by flash chromatography using ethylacetate as eluent to give 196 mg (27%) of the title compound.

(ii) H—(R)Cha-Pro-Pac(Z)

Hydrogen chloride was bubbled through a solution of 196 mgBoc-(R)Cha-Pro-Pac(Z) in 25 ml ethyl acetate. After 10 minutes thereaction mixture was diluted with methylene chloride and sodiumhydroxide solution was added. The aqueous phase was extracted severaltimes with methylene chloride and the combined organic phases were dried(K₂CO₃) and the solvent was removed in vacuo to give 86 mg (52%) of thetitle compound.

(iii) H—(R)Cha-Pro-Pac×2 HCl

The title compound was prepared by hydrogenation of H—(R)Cha-Pro-Pac(Z)in ethanol in the presence of 10% Pd/C.

H-NMR (300 MHz, D2O; A ca: 1:1 mixture of 1,4-cis- and 1,4-trans isomersin the Pac part of the molecule); δ 1.15-1.3 (q), 1.6-1.85 (m), 1.9-2.0(m), 2.0-2.1 (d), 2.1-2.15 (m), 2.15-2.2 (m), 2.65-2.7 (m), 2.7-2.8 (m),2.95-3.0 (d), 3.15-3.2 (d), 5.4 (s), 7.45-7.55 (m).

Example 54

N—(R)Cgl-Ile-Pab×2 HCl

(i) Boc-(R)Cgl-Ile-Pab(Z)

To a stirred mixture of 1.33 g (3.6 mmol) Boc-(R)Cgl-Ile-OH (SeePreparation of starting materials), 1.12 g (3.9 mmol) H-Pab(Z) (SeePreparation of starting materials) and 1.76 g (14.4 mmol) DMAP in 50 mlCH₃CN/DMF (1/1) was added 0.75 g (3.9 mmol) EDC at +5° C. The reactionmixture was allowed to reach room temperature and left for 60 h. TheCH₃CN was removed by evaporation and the residue was poured out in 100ml water (a yellow precipitate was formed). The mixture was extractedwith 2×50 ml EtOAc and the combined organic phase was washed with 2×30ml NaHCO₃(saturated), 2×50 ml 0.2 M HCl, 1×50 ml Brine and dried(MgSO₄). Evaporation followed by flash chromatography using CH₂Cl₂/THF(85/15) as eluent gave 510 mg (24%) of the title compound.

(ii) H—(R)Cgl-Ile-Pab(Z)

530 mg Boc-(R)Cgl-Ile-Pab(Z) was dissolved in 14 ml CH₂Cl₂/TFA (2.5/1)and stirred for 2 h at room temperature. Evaporation of the solventfollowed by flash chromatography using CH₂Cl₂/MeOH(NH₃-saturated) (95/5)as eluent gave the title compound.

(iii) H—(R)Cgl-Ile-Pab×2 HCl

75 mg (0.14 mmol) H—(R)Cgl-Ile-Pab(Z) was hydrogenated over 10% Pd/C in5 ml EtOH, which contained an excess HCl(g) to give the dihydrochloride,at atmospheric pressure for 6 h. Addition of 2 g activated charcoal and20 ml EtOH followed by filtration through celite, evaporation of thesolvent and freeze drying from water gave 50 mg (89%) of the titlecompound as a white powder.

¹H-NMR(500 MHz, MeOD): δ 0.90 (t, 3H), 0.94 (d, 3H), 1.1-2.0 (m, 14H),3.83 (bs, 1H), 4.26 (d, 1H), 4.50 (m, 2H), 7.57 (bd, 2H), 7.78 (bd, 2H).

Example 55

H—(R)Cgl-Aze-Pab

Hydrogenation of 257 mg (5.08 μmmol) H—(R)Cgl-Aze-Pab(Z) (See Example 1(ii)) over 5% Pd/C in 6 ml EtOH/H₂O at atmospheric pressure for 6 hfollowed by filtration of the catalyst, evaporation of the solvent andfreeze drying from water gave 200 mg (89%) of the title compound.

¹H-NMR (500 MHz, D₂O): δ 1.0-2.0 (m, 11H), 2.25 (m, 1H), 2.70 (m, 1H),3.30 (m, 1H), 3.75 (m, 1H), 4.30 (m, 1H), 4.45 (m, 1H), 4.55 (m, 2H),7.60 (m, 2H), 7.77 (m, 2H).

MS m/z 372 (M⁺+1).

Example 56

HOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab×HOAc

(i) BnOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab(Z)

0.250 g (0.47 mmol) H—(R)Cha-Pro-Pab(Z) (See Example 15) dissolved in 5ml CH₂Cl₂ was cooled to −10° C. and 150 mg (0.48 mmol) of TfOCH₂COOBn(See Preparation of Startingmaterials) dissolved in 3 ml CH₂Cl₂ wasadded slowly. 200 mg (1.45 mmol) of potassium carbonate was added andthe mixture was stirred at roomtemperature for 20 h. The mixture wasdiluted with CH₂Cl₂, extracted with water and dried (MgSO₄). Evaporationof the solvent followed by flash chromtography using CH₂Cl₂/MeOH 9/1 aseluent gave 150 mg (46%) of the title compound.

(ii) HOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab×HOAc

150 mg (0.2 mmol) BnOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab(Z) was hydrogenatedover 50 mg 5% Pd/C in 20 ml EtOH at athmospheric pressure for 4 h.Filtration of the catalyst, evaporation of the solvent followed bypurification by RPLC, using CH₃CN/0.1 M NH₄OAc 1/4 as eluent, gave 35 mg(37%) of the title compound.

¹H-NMR (500 MHz, MeOD): δ 1.00 (m, 1H), 1.20-1.45 (m, 5H), 1.5 (m, 1H),1.6-1.8 (m, 6H), 1.9-2.1 (m, 6H), 2.25 (m, 1H), 3.25 (m, 1H), 3.5 (m,1H), 3.85 (m, 1H), 4.15 (m, 1H), 4.35-4.6 (m, 3H), 4.9 (m, partiallyhidden by the HOD line, 6H), 7.55 (d, 2H), 7.75 (d, 2H).

Example 57

MeOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

(i) MeOOC—CH₂—(R)Cgl-Aze-Pab(Z)

0.186 g (0.841 mmol) TfO-CH₂—COOMe (See preparation of startingmaterials) was dissolved in CH₂Cl₂ and slowly added to a mixture of0.425 g (0.841 mmol) H—(R)Cgl-Aze-Pab(Z) (See Example 1), 0.894 g (5.04mmol) K₂CO₃ in CH₂Cl₂ (totally 4.3 ml) at roomtemperature, and stirredover night. More CH₂Cl₂ was added and the mixture was washed with waterand brine, dried, filtered and the solvent was evaporated in vacuo togive 0.51 g of a residue that was three times subjected to flashchromatography on silica gel using, first CH₂Cl₂/THF/MeOH (16/4/1), thenCH₂Cl₂/THF(2% NH₃) (8/2) and the last timediethylether/MeOH(NH₃-saturated) (95/5) as eluent. This gave 0.324 g(67%) of the title compound.

(ii) MeOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

220 mg (0.38 mmol) MeOOC—CH₂—(R)Cgl-Aze-Pab(Z) was hydrogenated in thepresence of 1.14 ml 1 N HCl, 6.5 ml MeOH and 300 mg Pd/C for 2 h.Removal of the catalyst by filtration on cellite and millipore filterfollowed by evaporation of the solvent in vacuo and freeze drying twicegave 178 mg (91%) of the title compound.

¹H-NMR (500 MHz, D₂O); δ 1.12-1.4 (m, 5H), 1.68-1.81 (m, 2H), 1.81-1.9(m, 3H), 1.97-2.1 (m, 1H), 2.29-2.4 (m, 1H), 2.68-2.8 (m, 1H), 3.86 (s,3H), 4.1 (s, 2H), 4.1-4.5 (d, 1H), 4.36-4.42 (t, 2H), 4.59 (s, 2H),4.99-5.04 (m, 1H), 7.65-7.7 (d, 2H), 7.8-7.85 (d, 2H).

¹³C-NMR (75 MHz, MeOD): amidine and carbonyl carbons; δ 146.78, 167.68,168.15, 172.29.

Example 58

EtOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

(i) EtOOC—CH₂—(R)Cgl-Aze-Pab(Z)

0.208 g (0.876 mmol) TfO-CH₂—COOEt (See preparation of startingmaterials) was dissolved in CH₂Cl₂ and slowly added to a mixture of0.443 g (0.876 mmol) H—(R)Cgl-Aze-Pab(Z) (See Example 1) and 0.931 g(5.26 mmol) K₂CO₃ in CH₂Cl₂ (totally 4 ml) cooled on an ice-bath. After2 h the ice-bath was removed and stirring was continued atroomtemperature for 2 hours. More CH₂Cl₂ was added and the mixture waswashed with water and brine, dried, filtered and the solvent wasevaporated in vacuo to give 0.51 g of a residue that was subjected toflash chromatography using diethylether/MeOH(NH₃-saturated) (95/5) aseluent. This gave 0.387 g (75%) of the title compound.

(ii) EtOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

395 mg (0.668 mmol) EtOOC—CH₂—(R)Cgl-Aze-Pab(Z) was hydrogenated in thepresence of 12 ml EtOH (99.5%) and 390 mg Pd/C for 5 h. Removal of thecatalyst by filtration on cellite and millipore filter, followed byevaporation of the solvent in vacuo and freeze drying twice, gave 281 mg(88%) of EtOOC—CH₂—(R)Cgl-Aze-Pab. 2 eqvivalents of 1 N HCl was added,and freeze drying three times gave 288 mg (81%) of the title compound.

¹H-NMR (500 MHz, D₂O); δ 1.05-1.48 (m, 8H), 1.6-2.05 (m, 6H), 2.15-2.33(m, 1H), 2.58-2.79 (m, 1H), 3.89-4.0 (m, 3H), 4.2-4.33 (m, 3H),4.33-4.44 (m, 1H), 4.44-4.66 (m, 2H), 4.91 (m, 1H (partially hidden bythe H-O-D signal)), 7.54-7.63 (d, 2H), 7.72-7.84 (d, 2H).

Example 59

^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab×HOAc

(i) ^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab(z)

Prepared in the same way as described for^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab(Z) (See Example 60 (i)) usingTfO-CH₂—COO^(n)Bu as alkylating agent. The crude product was purified byflash chromatography twice, first using CH₂Cl₂/MeOH (95/1) as eluent andthen CH₂Cl₂/i-propylalcohol (90/7) to give 324 mg (47%) of the titlecompound.

(ii) ^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab×HOAc

The deprotection was done according to the procedure described inExample 57 (ii). The crude material was purified on RPLC using CH₃CN(30%) in 0.05 M NH₄OAc and 0.05 M HOAc as eluent to give 100 mg (53%) ofthe title compound.

¹H-NMR (300 MHz, MeOD); δ 0.85-2.1 (m, 18H), 2.15-2.37 (m, 1H), 2.58-2.8(m, 1H), 3.7-5.0 (m, 10H), 4.88-5.0 (partially hidden by the H-O-Dsignal)), 7.46-7.65 (d, 2H), 7.71-7.88 (d, 2H).

¹³C-NMR (75 MHz, MeOD): amidine and carbonyl carbons; δ 146.8, 168.12,168.2, 172.2.

Example 60

^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

(i) ^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab(Z)

0.402 g (1.375 mmol) TfO-CH₂—COO^(n)Hex (See Preparation of startingmaterials) was dissolved in CH₂Cl₂ and slowly added to a mixture of0.695 g (1.375 mmol) H—(R)Cgl-Aze-Pab(Z) (See Example 1), 1.463 g (8.25mmol) K₂CO₃ in CH₂Cl₂ (totally 4 ml) at <−10° C. After 1 h theCO₂-ice-bath was removed and stirring was continued at room temperaturefor 45 minutes. More CH₂Cl₂ was added and the mixture was washed withwater and brine, dried, filtered and the solvent was evaporated in vacuoto give 0.828 g of a residue, wich was twice subjected to flashchromatography, first using diethylether/MeOH(NH₃-saturated) (95/5), andthen CH₂Cl₂/MeOH(NH₃-saturated) (95/5) as eluent. This gave 0.42 g (47%)of the title compound.

(ii)_(n)HexOOC—CH₂—(R)Cgl-Aze-Pab×2 HCl

Hydrogenation of 400 mg (0.617 mmol)_(n)HexOOC—CH₂—(R)Cgl-Aze-Pab(Z) inthe presence of 12 ml THF and 400 mg Pd/C for 1.5 h did not givecomplete de-protection. The hydrogenation was completed in 4 h in thepresence of 1.7 ml 1 N HCl, 12 ml MeOH and 340 mg Pd/C. Removal of thecatalyst by filtration on cellite and millipore filter, followed byevaporation of the solvent in vacuo and freeze drying twice, gave 287 mg(79%) of the title compound.

¹H-NMR (300 MHz, MeOD); δ 0.8-2.13 (m, 22H), 2.13-2.31 (m, 1H),2.61-2.81 (m, 1H), 3.93-4.15 (m, 3H), 4.15-4.37 (m, 3H), 4.37-4.7 (m,3H), 4.88-5.0 (m, 1H (partialyy hidden by the H-O-D signal)), 7.52-7.69(d, 2H), 7.75-7.9 (d, 2H).

¹³C-NMR (75 MHz, MeOD): amidine and carbonyl carbons; δ 146.84, 167.67,167.84, 172.17.

Example 61

H—(R)Cgl-Pro-Pac×2 HCl

(i) Boc-(R)Cgl-Pro-Pac(Z)

377 mg (1.97 mmol) EDC was added at 0° C. to a stirred solution of 708mg (1.95 mmol) of Boc-(R)Cgl-Pro-OH (See Preparation ofstartingmaterial), 714 mg (2.0 mmol) H-Pac(Z)×2 HCl (See Preparation ofstartingmaterial) and 1.078 g (8.8 mmol) DMAP in 12.5 ml acetonitrile.The reaction mixture was allowed to reach room temperature over night.The solvent was removed in vacuo and the residue was first purified byflash chromatography, using 10% methanol in methylene chloride aseluent, and subsequently by RPLC. Two fractions (51 mg and 150 mg)giving MS m/z=626 (M+1) were isolated.

(ii) H—(R)Cgl-Pro-Pac(Z)

Hydrogen chloride was bubbled into a solution of 141 mg (0.22 mmol)Boc-(R)Cgl-Pro-Pac(Z) in 50 ml ethyl acetate. After 15 minutes 10%sodium carbonate solution was added and the organic phase was separatedand dried (K₂C₃). Evaporation of the solvent gave 71 mg (61%) of theproduct.

(iii) H—(R)Cgl-Pro-Pac×2 HCl

A mixture of 71 mg (0.14 mmol) H—(R)Cgl-Pro-Pac(Z) and a small spatulaof 10% Pd/C in 10 ml of ethanol was hydrogenated at room temperature andatmospheric pressure for 2 h. The catalyst was removed by filtration andthe solvent was removed in vacuo. The residue was dissolved in 50 mlwater and 0.6 g 1M hydrochloric acid. Freeze drying yielded 38 mg (58%)of the title compound.

MS m/z 392 (M+1)

Example 62

HOOC—CH₂—(R)Cha-Pro-Pac×HOAc

(i) BnOOC—CH₂—(R)Cha-Pro-Pac(Z)

A mixture of 84 mg (0.15 mmol) H—(R)Cha-Pro-Pac(Z) (See Example 53(ii)), one spatula of potassium carbonate, and 47 mg of TfOCH₂—COOBn(See Preparation of starting materials) in 3 ml of methylene chloridewas stirred at room temperature over night. The reaction mixture wasfiltered and the solvent was removed in vacuo to give a residue whichwas subjected to flash chromatography using ethyl acetate/methylenechloride/methanol 95:20:5 as eluent. 29 mg of the desired product wasisolated.

(ii) HOOC—CH₂—(R)Cha-Pro-Pac×HOAc

A mixture of 29 mg BnOOC—CH₂—(R)Cha-Pro-Pac(Z) and 37 mg of 10% Pd—C in5 ml etanol was stirred for 4 h at room temperature and atmosphericpressure. Filtration of the catalyst followed by removal of the solventand purification by RPLC gave the desired compound.

MS m/z 464 (M+1).

Example 63

HOOC—CH₂—CH₂—(R)Cgl-Pro-Pac

(i) BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pac(Z)

A solution of 0.35 g (0.64 mmol) H—(R)Cgl-Pro-Pac(Z) (See Example 61(ii)), 124 mg (0.76 mmol) benzyl acrylate, and 280 μl (2 mmol) triethylamine in 1 ml ethanol was kept at room temperature for 3 days. Removalof the solvent followed by purification by HPLC gave 18 mg (4%) of thetitle compound.

(ii) HOOC—CH₂—CH₂—(R)Cgl-Pro-Pac

A mixture of 18 mg BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pac(Z) and a small spatulaof 10% Pd/C was hydrogenated for 2 h at room temperature and atmosphericpressure in EtOH. Filtration followed by removal of the solvent in vacuoand dissolution in water and freeze drying gave 7 mg (78%) of the titlecompound. MS m/z 464 (M+1).

Example 64

HOOC—CH₂—CH₂—(R)Cha-Aze-Pac

(i) Boc-(R)Cha-Aze-Pac(Z)

A solution of 0.4 g (1.38 mmol) H-Pac(Z) (See Preparation ofstartingmaterial of H-Pac(Z)×2 HCl), 0.5 g (1.41 mmol)Boc-(R)Cha-Aze-OH(See Preparation of startingmaterial), and 0.67 g (5.5mmol) DMAP in 20 ml acetonitrile was mixed at 0° C. with a solution of0.26 g (1.4 mmol) EDC in 15 ml acetonitrile. The reaction mixture waskept at room temperature over night and the solvent was subsequentlyremoved in vacuo. The residue was partitioned between ethyl acetate andwater. The aqueous phase was extracted once more with ethyl acetate andthe combined organic phases were washed with sodium hydrogen sulphatesolution, sodium carbonate solution, and brine and then dried (sodiumsulphate). Evaporation of the solvent gave 0.54 g (63%) of the titlecompound.

(ii) H—(R)Cha-Aze-Pac(Z)

Hydrogen chloride was bubbled into a solution of 0.54 g (0.9 mmol)Boc-(R)Cha-Aze-Pac(Z) in ethyl acetate. The solution was kept in therefrigerator over night and the solvent was then removed in vacuo andthe residue was dissolved in ethyl acetate. The solution was washed withaqueous sodium hydrogen carbonate solution, water and brine and dried(sodium sulphate). Removal of the solvent gave 0.35 g (77%) of theproduct.

(iii) BnOOC—CH₂—CH₂—(R)Cha-Aze-Pac(Z)

A solution of 180 mg (0.33 mmol) H—(R)Cha-Aze-Pac(Z) and 53 mg (0.33mmol) benzyl acrylate in ethanol was kept at room temperature for 60 h.The solvent was removed in vacuo and the residue was dissolved in ethylacetate. The solution was washed with potassium hydrogen sulphatesolution and sodium hydrogen carbonate solution and brine. Drying(sodium sulphate) and removal of the solvent in vacuo gave a residuewhich was purified by flash chromatography, using 10% methanol inmethylene chloride as eluent to yield 150 mg (66%) of the title compound

(iv) HOOC—CH₂—CH₂—(R)Cha-Aze-Pac×2 HCl

A mixture of 115 mg BnOOC—CH₂—CH₂—(R)Cha-Aze-Pac(Z) and 67 mg of 10%Pd—C in 10 ml ethanol was hydrogenated for 1.5 h at room temperature andatmospheric pressure. Filtration followed by removal of the solvent invacuo and dissolution of the residue in water and 1.5 ml of 1Mhydrochloric acid gave a solution which was freeze dried to give 30 mg(33%) of the title compound.

MS m/z 464 (M+1).

Example 65

HOOC—CH₂—(R)cha-Aze-Pig×2 HCl

(i) BOC-(R)cha-Aze-Pig(z)

0.249 g (1.298 mmol) EDC was added at <−15° C. to a mixture of 0.473 g(1.236 mmol) Boc-(R)Cha-Aze-OH (See Preparation of starting materials),0.404 g (1.236 mmol) H-Pig(Z)×HCl (See Preparation of startingmaterials) and 0.604 g (4.94 mmol) DMAP in 13.5 ml DMF. The temperaturewas allowed to rise to roomtemperature over night. The solvent wasevaporated in vacuo and EtOAc and 2 M KHSO₄ was added. The phases wereseparated and the organic phase was washed with saturated Na₂CO₃ andbrine. Repeting the extractive procedure, drying (Na₂SO₄), filtrationand evaporation of the solvents gave 0.612 g of a residue which wassubjected to flash chromatography using EtOAc/MeOH 9/1 as eluent. Thisgave 407 mg (53%) of the title compound.

(ii) H—(R)cha-Aze-Pig(z)

0.4 g (0.638 mmol) of Boc-(R)Cha-Aze-Pig(Z) was dissolved in 24.4 ml ofTFA/CH₂Cl₂ 1/4, stirred for 30 minutes on an ice-bath, and for 30minutes at roomtemperature. The solvent was removed in vacuo and EtOAcand saturated Na₂CO₃ was added. The phases were seperated and theorganic layer was washed with water and brine, dried (Na₂SO₄), filteredand the solvent was evaporated in vacuo to give 336 mg (100%) of thetitle compound.

(iii) BnOOC—CH₂—(R)Cha-Aze-Pig(Z)

89 ml (0.562 mmol) BnOOC—CH₂—Br was slowly added to a mixture of 0.296 g(0.562 mmol) H—(R)Cha-Aze-Pig(Z) and 0.171 g (1.236 mmol) K₂CO₃ in 6 mlCH₃CN heated to 60° C. on an oilbath. After 1 h 45 minutes the solventwas evaporated, EtOAc was added, and the mixture was washed with water,dried (Na₂SO₄), filtered and the solvent was evaporated in vacuo to give346 mg of a residue which was subjected to flash chromatography usingCH₂Cl₂/THF/MeOH (8/2/1) as eluent. This gave 297 mg (78%) of the titlecompound.

(iv) HOOC—CH₂—(R)Cha-Aze-Pig×2 HCl

243 mg (0.36 mmol) BnOOC—CH₂—(R)Cha-Aze-Pig(z) was hydrogenated in thepresence of 1.7 ml 1 N HCl, 10 ml EtOH (99.5%) and 300 mg Pd/C for 2 h.Removal of the catalyst by filtration on cellite and millipore filterfollowed by evaporation of the solvent in vacuo and freeze drying twicegave 166 mg (88%) of the title compound.

¹H-NMR (500 MHz, D₂O); δ 0.6-1.9 (m, 18H), 2.1-2.27 (m, 1H), 2.52-2.76(m, 1H), 2.82-3.2 (m, 4H), 3.46-3.61 (m, 1H), 3.61-3.81 (m, 2H),3.81-4.0 (m, 2H), 4.0-4.24 (m, 2H), 4.24-4.4 (m, 1H).

Example 66

HOOC—CH₂—(R)Cha-Pro-Pig×2 HCl

(i) Boc-(R)Cha-Pro-Pig(Z)

To a mixture of 0.3495 g (0.95 mmole) Boc-(R)Cha-Pro-OH (see Preparationof starting materials), 0.464 g (3.8 mmole) DMAP, 0.310 g (0.95 mmole)H-Pig(Z)×HCl (See Preparation of starting materials) in 5 ml CH₂Cl₂ wasadded 0.192 g (1 mmole) of EDC and the mixture was stirred over night atroom temperature. The mixture was evaporated and the residue wasdissolved in ethyl acetate. The organic phase was washed twice with 0.3M KHSO₄ and once with brine. The organic layer was dried (Na₂SO₄),filtered and evaporated. The crude product was purified by flashchromatography using a stepwise gradient of CH₂Cl₂/MeOH (100/0, 97/3,95/5, 90/10) as eluent to yield 307 mg of the title compound.

(ii) H—(R)Cha-Pro-Pig(Z)

0.306 g (0.48 mmole) Boc-(R)Cha-Pro-Pig(Z) was dissolved in 30 ml HClsaturated ethyl acetate. The mixture was allowed to stand for half anhour. The solvent was evaporated and the residue was dissolved inCH₂Cl₂. The organic layer was washed twice with 0.2 M NaOH. The combinedwater layer was extracted once with CH₂Cl₂ and the combined organiclayer was dried (Na₂SO₄), filtered and evaporated to yield 257 mg (99%)of the title compound.

(iii) BnOOC—CH₂—(R)cha-Pro-Pig(z)

A mixture of 0.256 g (0.473 mmole) H—(R)Cha-Pro-Pig(Z), 0.144 g (1.04mmole) K₂CO₃ and 82 μl (0.521 mmole) of bensylbromoacetate in 6 mlacetonitrile was heated to 60° C. for two hours under stirring. Thesolvent was evaporated and the residue was dissolved in CH₂Cl₂, washedonce with water and once with brine, dried (Na₂SO₄), filtered and thesolvent evaporated. The crude product was purified by flashchromatography using a stepwise gradient of CH₂Cl₂/MeOH (97/3, 95/5,90/10) as eluent to yield 0.2 g product (90% pure according to RPLC).The final purification was made on a chromatotron (Harrison research,model 7924T) on a 2 mm silica plate in CH₂Cl₂/MeOH 95/5 yielding 0.158 q(48%) of the pure product.

(iv) HOOC—CH₂—(R)Cha-Pro-Pig×2 HCl

0.158 g (0.227 mmole) of BnOOC—CH₂—(R)Cha-Pre-Pig(Z) was mixed with0.075 g Pd/C (5%), 1.0 ml 1N HCl-solution and 10 ml ethanol. The mixturewas hydrogenated at atmospheric for one hour. Filtration through celliteand evaporation of the solvent followed by freeze drying twice fromwater gave 119 mg (97%) of the product.

¹H-NMR (D2O, 300 MHz): δ 0.95-1.44 (m, 7H), 1.52 (m, 1H), 1.60-2.20 (m,13H), 2.39 (m, 1H), 3.07-3.32 (m, 4H), 3.68 (m, 1H), 3.77-4.02 (m, 5H;thereof 3.98 (s, 2H), 4.44-4.58 (m, 2H)

¹³C-NMR (D₂O, 75 MHz): carbonyl- and guanidine carbons: δ 156.5, 168.3,169.6, 174.5

Example 67

HOOC—CH₂—CH₂—(R)Cha-Pro-Pig×2 HCl

(i) BnOOC—CH₂—CH₂—(R)Cha-Pro-Pig(Z)

0.297 g (0.55 mmole) H—(R)Cha-Pro-Pig(Z) (See Example 66 (ii)) wasdissolved in 2 ml ethanol and 90 μl (0.59 mmole) bensylacrylate wasadded. The reaction mixture was stirred for four days at roomtemperature. The solvent was evaporated and the crude productchromatographed on a chromatotron (Harrison research, model 7924T) usinga 2 mm silica plate with a stepwise gradient of CH₂Cl₂/MeOH (95/5,90/10) as eluent to yield 0.338 g (87%) of the title compound.

(ii) HOOC—CH₂—CH₂—(R)Cha-Pro-Pig×2 HCl

0.238 g (0.227 mmole) of BnOOC—CH₂—CH₂—(R)Cha-Pro-Pig(Z) was mixed with0.120 g Pd/c (5%), 1.2 ml 1N HCl-solution and 15 ml ethanol. The mixturewas hydrogenated at atmospheric pressure for one hour. Filtration of thecatalyst through cellite, evaporation of the solvent followed by freezedrying twice from water gave 178 mg (95%) of the title compound.

¹H-NMR (D₂O, 300 MHz): δ 0.82-1.45 (m, 5H), 1.45-2.15 (m, 13H), 2.29 (m,1H), 2.83 (t, 2H), 2.9-3.4 (m, 6H), 3.57 (bq, 1H), 3.67-3.87 (m, 3H),4.25-4.43 (m, 2H)

¹³C-NMR (D₂O, 75 MHz): carbonyl- and guanidine carbons: δ 156.3, 168.2,174.3, 174.6

MS m/z 479 (M⁺+1)

Example 68

(HOOC—CH₂)₂—(R)Cgl-Pro-Pig×2 HCl

120 mg (0.126) mmol (BnOOC—CH₂)₂—(R)Cgl-Pro-Pig(Z)₂ (See Example 50(iii)) was hydrogenated in the presence of 0.75 ml 1 N HCl, 7 ml EtOH(99.5%) and 150 mg Pd/C for 4 h. Removal of the catalyst by filtrationon cellite and millipore filter and evaporation of the solvent in vacuofollowed by freeze drying gave 66 mg (90%) of the title compound.

¹-NMR (500 MHz, D₂O); δ 1.05-1.38 (m, 7H), 1.53-1.64 (d, 1H), 1.64-2.14(m, 11H), 2.27-2.39 (m, 1H), 3.03-3.28 (m, 4H), 3.58-3.70 (m, 1H),3.7-3.8 (m, 1H), 3.8-3.9 (d, 2H), 4.07-4.22 (m, 2H), 4.22-4.35 (m, 1H),4.38-4.5 (m, 1H).

¹³C-NMR (75 MHz, D₂O): amidine and carbonyl carbons; δ 156.28, 166.73,170.14, 174.01.

Example 69

HOOC—CH₂—CH₂—(HOOC—CH₂)—(R)Cha-Pro-Pig×2 HCl

(i) BnOOC—CH₂—CH₂-(BnOOC—CH₂)—(R)Cha-Pro-Pig(Z)

To a cold (ice-bath temperature) mixture of 100 mg (0.14 mmol)BnOOC—CH₂—CH₂—(R)Cha-Pro-Pig(Z) (See Example 67 (i)) and 80 mg (0.57mmol) potassium carbonate in 4 ml of CH₂Cl₂ was carfully added asolution of 64 mg (0.21 mmol) TfO-CH₂—COOBn dissolved in 1 ml CH₂Cl₂.The reaction mixture was left at 0° C. for 30 minutes and then allowedto reach room temperature for 2 h after which it was heated to refluxfor 30 minutes and finally left over night at room temperature.Evaporation of the solvent followed by flash chromatography usingCH₂Cl₂/MeOH (97/3) as eluent afforded 65 mg (54%) of the title compound.

(ii) HOOC—CH₂—CH₂—(HOOC—CH₂)—(R)Cha-Pro-Pig×2 HCl

65 mg (0.08 mmol) of BnOOC—CH₂—CH₂-(BnOOC—CH₂)—(R)Cha-Pro-Pig(Z) wasdissolved in 10 ml of EtOH/1M HCl (9/1) and hydrogenated over 10% Pd/Cfor 3 h at athmospheric pressure. Filtration of the catalyst evaporationof the solvent followed by freeze drying from water gave 40 mg (97%) ofthe title compound as a white powder.

¹³C-NMR (125 MHz, MeOD): amidine and carbonyl carbons: δ 157.5, 167.2,169.1, 173.7 and 174.1.

Example 70

HOOC—CH₂—(R)Cgl-Aza-(R,S)Itp×2 HCl

(i) Boc-(R)Cgl-Aze-(R,S)Itp(Ts)

Boc-(R)Cgl-Aze-OH (See Preparation of starting materials) (400 mg, 1.17mmol), H—(R,S)Itp(Ts) (See Preparation of starting materials) (366 mg,1.23 mmol) and DMAP (286 mg, 2.34 mmol) was dissolved in CH₃CN (6 ml)and cooled to 5° C. EDC (236 mg, 1.23 mmol) was added and the resultingmixture was stirred at room temperature over night. The CH₃CN wasremoved and the residue was disolved in MeOH/EtOAc/H₂O. The separatedorganic layer was washed with K₂CO₃(sat), 2 M KHSO4, brine anddried(Na₂SO₄). Evaporation of the solvent resulted in a white solid, 688mg (85%).

MS m/z 620 (M⁺+1)

(ii) H—(R)Cgl-Aze-(R,S)Itp(Ts)

Boc-(R)Cgl-Aze-(R,S)Itp(Ts) (500 mg, 0.8 mmol) was dissolved in CH₂Cl₂(50 ml) and HCl(g) was bubbled through the solution for ca 4 min. After45 min the solvent was removed by evaporation and the resulting productwas dissolved in EtOAc/MeOH/H₂O and the acidic solution was treated with2 N NaOH(aq) to pH=8-9. The organic layer was separated anddried(Na₂SO₄). Evaporation of the solvent afforded 425 mg (100%) of thetitle compound as a white solid.

MS m/z 520 (M⁺+1)

(iii) BnOOC—CH₂—(R)Cgl-Aze-(R,S)Itp(Ts)

H—(R)Cgl-Aze-(R,S)Itp(Ts) (400 mg, 0.77 mmol),Benzyl-2-(para-nitrobenzenesulfonyloxy)acetate (See Preparation ofstarting materials) (325 mg, 0.92 mmol) and K₂CO₃ (235 mg, 1.7 mmol) wasstirred in CH₃CN (5 ml) at 45° C. After a few hours the conversion wasonly 25% and therefore the temperature was increased to 60° C. and anadditional amount of Benzyl-2-(para-nitrobenzenesulfonyloxy)acetate wasadded.

The reaction was stirred for 48 h, (startingm.:product/25:63), and thenworked up. The solvent was evaporated and EtOAc/H₂O was added to theresidue. The phases were separated and the water-phase was washed twicewith EtOAc and then the combined organic phase was washed withK₂CO₃(sat), 2 M KHSO4, H₂O and dried Na₂SO₄). This aforded, afterback-extraction of the acidic KHSO₄, some 340 mg which was purified byRPLC. This gave 34 mg (7%) of the title compound.

MS m/z 668 (M⁺+1).

(iv) HOOC—CH₂—(R)Cgl-Aze-(R,S)Itp×2 HCl

BnOOC—CH₂—(R)Cgl-Aze-(R,S)Itp(Ts) (34 mg, 0.05 mmol) was dissolved inTHF (5 ml) and NH₃(g) was destined (40 ml) into the reaction flask witha dry-ice cooler. Na(s) was added and a deep blue color appeared. Thereaction was stirred for 5 min before it was quenched with HOAc (50 μl).The dry-ice cooler was removed and the NH₃(l) was allowed to evaporate.To the residue H₂O and HOAc was added to pH=7. Freeze-drying andpreparative RPLC gave several fractions which were analyzed with FAB-MS.Two fractions contained the desired compound, 3 mg (10%) afterfreeze-drying with 2.2 eq of 1 M HCl:

MS m/z 424 (M⁺+1).

Example 71

HOOC—CH₂—(R)Cha-Aze-(R,S)Itp

(i) Boc-(R)Cha-Aze-(R,S)Itp(Ts)

Boc-(R)Cha-Aze-OH (See Preparation of starting materials) (169 mg, 0.5mmol), H—(R,S)Itp(Ts) (See Preparation of starting materials) (155 mg,0.52 mmol), DMAP (122 mg, 1 mmol) was dissolved in CH₃CN (2.5 ml) andcooled to 5° C. EDC×HCl (115 mg, 0.6 mmol) was added and the resultingmixture was stirred at room temperature over night. Extra (0.5 eq)H—(R,S)Itp(Ts) and EDC was added after stirring over nigth. The reactionmixture was stirred an additional night and worked up as described inthe Boc-(R)Cgl-Aze-(R,S)Itp(Ts) (See Example 70) case above. This gave260 mg of crude product. Purification by RPLC gave 180 mg (57%) of thetitle compound.

MS m/z 634 (M⁺+1).

(ii) H—(R)Cha-Aze-(R,S)Itp(Ts)

Boc-(R)Cha-Aze-(R,S)Itp(Ts) (180 mg, 0.28 mmol) was dissolved in CH₂Cl₂(20 ml) and HCl(g) was bubbled through the solution for ca 4 min. After45 min the solvent was removed by evaporation and the resulting productwas dissolved in CH₂Cl₂ and washed with 2 M NaOH to pH=8-9. The phaseswere separated and the organic phase was dried(Na₂SO₄) and evaporated toyield 163 mg (ca 100%):

MS m/z 534 (M⁺+1).

(iii) BnOOC—CH₂—(R)Cha-Aze-(R,S)Itp(Ts)

H—(R)Cha-Aze-(R,S)Itp(Ts) (80 mg, 0.15 mmol), K₂CO₃ (45 mg, 0.33 mmol)and Br—CH₂COOBn (39 mg, 0.17 mmol) was stirred in CH₃CN (1.5 ml) at 60°C. for 2.5 h. The solvent was evaporated and the residue was dissolvedin EtOAc/H₂O. The phases were separated and organic phase was washedwith 10% citric acid and dried (Na₂SO₄). Evaporation of the solvent gavea 171 mg of crude product, which was purified by RPLC yielding 53 mg(52%) of the title compound.

MS M/z 681 (M⁺+1).

(iv) HOOC—CH₂—(R)Cha-Aze-(R,S)Itp

BnOOC—CH₂—(R)Cha-Aze-(R,S)Itp(Ts) (50 mg, 0.07 mmol) was treated asdescribed for BnOOC—CH₂—(R)Cgl-Aze-(R,S)Itp(Ts) (See Example 70 (iv)).This gave a product mixture which was purified on a RPLC yielding 12 mgof a 1:1 mixture of the title compound together with a reduced formwhich appear at mass 439 (m/z).

MS m/z 438 (M⁺+1)

Example 72

H—(R)Cha-Pic-(R,S)Itp×2 HCl

(i) Boc-(R)Cha-Pic-(R,S)Itp(Ts)

At roomtemperature 2.1 g (5.5 mmol) Boc-(R)Cha-Pic-OH (See Preparationof starting materials), 1.0 g (8.2 mmol) DMAP and 1.7 g (5.8 mmol)H—(R,S)Itp(Ts) (See Preparation of starting materials) was dissolved in40 mL acetonitrile. After a few minutes of stirring 1.1 g (5.8 mmol) EDCwas added and the stirring was continued for 60 hours. The solvent wasremoved in vacuo and the residue was dissolved in CH₂Cl₂, washed withwater, 0.3M KHSO₄ and KHCO3 (aq) and dried(Na₂SO₄). Evaporation of thesolvent and filtration through Silica gel gave 2.43 g (67%) of theproduct.

MS m/z 661 (M⁺+1)

(ii) Boc-(R)Cha-Pic-(R,S)Itp

2.4 g (3.6 mmol) Boc-(R)Cha-Pic-(R,S)Itp(Ts) was dissolved in 15 mL THFand NH₃ (g) was condensed into the flask followed by addition of Na. Thereaction was quenched after 5 min with acetic acid and the NH₃ and theTHF was evaporated. The residue was freezedried from water and purifiedby RPLC (CH₃CN/0.1M NH₄OAc, 6/4) to give 0.93 g (51%) of the desiredproduct.

MS m/z 507 (M⁺+1)

(iii) H—(R)Cha-Pic-(R,S)Itp×2 HCl

At roomtemperature 50 mg (0.099 mmol) Boc-(R)Cha-Pic-(R,S)Itp wasdissolved in ethylacetate saturated with HCl

(g). After stirring 2 h the solvent was removed in vacuo. The residuewas freezedried from water three times to give 35 mg (74%) of thedesired product.

MS m/z 407 (M⁺+1)

Example 73

HOOC—CH₂—(R)Cha-Pic-(R,S)Itp×2 HCl

(i) Boc-(R)Cha-Pic-(R,S)Itp(Z)

At roomtemperature 0.84 g (1.66 mmol) Boc-(R)Cha-Pic-(R,S)Itp (SeeExample 72) was dissolved in 10 mL CH₂Cl₂ and 10 mL 0.5M NaOH. 0.29 μL(1.82 mmol) Z-Cl was added dropwise. After stirring for 3 h the phaseswas separated and the organic phase was washed with water and dried overNa₂SO₄. Evaporation and flash chromatography (ethylacetate/heptane 9/1)gave 0.5 g (47%) of the desired product.

MS m/z 641 (M⁺+1)

(ii) H—(R)Cha-Pic-(R,S)Itp(Z)

At roomtemperature 0.5 g (0.78 mmol) Boc-(R)Cha-Pic-(R,S)Itp(Z) wasdissolved in ethylacetate saturated with HCl. Water was added and themixture was made basic with K₂CO₃. The phasees was separated. Thewaterphase was extracted with CH₂Cl₂ and the organic phase was washedwith water. The combined organic phase was then dried(Na₂SO₄).Evaporation of the solvent gave 0.3 g (71%) of the desired product.

MS m/z 541 (M⁺+1)

(iii) BnOOC—CH₂—(R)Cha-Pic-(R,S)Itp(Z)

0.29 g (0.5 mmol) H—(R)Cha-Pic-(R,S)Itp(Z), 0.15 g (1 mmol) K₂CO₃ wastaken up in 25 mL acetonitrile. 154 mg (0.6 mmol) benzylbromoacetate wasadded and the mixture was stirred at 50° C. for 4 h. evaporation andpurification by RPLC (acetonitrile:0.1M NH₄OAc 70:30) gave about 200 mgof the desired product.

(iv) HOOC—CH₂—(R)Cha-Pic-(R,S)Itp×2 HCl

200 mg BnOOC—CH₂—(R)Cha-Pic-(R,S)Itp(Z) was dissolved in ethanol. Asmall spoon of lot Pd on charcoal was added and the mixture washydrogenated for 4 h. Filtration through hyflo, evaporation of thesolvent followed by freezedrying from water gave 53 mg of the desiredproduct.

¹H NMR (300.13 MHz, D₂O); δ 1.0-2.35 (overlapping m, 22H), 3.28-3.51 (M,5H), 3.51-3.64 (m, 1H), 3.75-4-03 (m, 3H), 5.03-5.14 (s broad, 1H). Thesignal of one of the protons is partially obscured by the H-O-D-signal.

MS m/z 465 (M⁺+1)

Example 74

H—(R)Cgl-Pro-(R,S)Hig×2 HCl

(i) Boc-(R)Cgl-Pro-(R,S)Hig(Z)

To a mixture of 1.0 g (2.95 mmole) Boc-(R)Cgl-Pro-OH (See Preparation ofstartingmaterials), 1.44 g (11.8 mmole) DMAP, 1.12 g (3.25 mmole)H—(R,S)Hig(Z) (See Preparation of startingmaterials) in 15 ml CH₂Cl₂ wasadded 0.62 g (3.2 mmole) of EDC and the mixture was stirred at roomtemperature over night. The solvent was evaporated and the residue wasdissolved in ethyl acetate. When the organic layer was washed twice witha 0.3 M KHSO₄-solution an oil separated from the organic layer. Theethyl acetate layer was dried (Na₂SO₄) and filtered. The oil and thewater layer was then extracted with CH₂Cl₂ The organic layer was dried(Na₂SO₄), filtered and combined with the EtOAc phase from above.Evaporation and purification of the crude product on a chromatotron(Harrison research, model 7924T) using a 2 mm silica plate with astepwise gradient of CH₂Cl₂/MeOH (97/3, 95/5, 90/10 as eluent to yielded1.1 g (59%) of the title compound.

(ii) H—(R)Cgl-Pro-(R,S)Hig×2 HCl

81 mg (0.13 mmole) of Boc-(R)Cgl-Pro-(R,S)Hig(Z) was dissolved in 50 mlethyl acetate saturated with HCl.

The mixture was allowed to stand for one hour, evaporated and theresidue was dissolved in 10 ml ethanol. 40 mg Pd/C (5%), 1 ml water and0.5 ml 1 M HCl-solution was added and the mixture was hydrogenated atatmospheric pressure over night. Filtration of the catalyst throughcellite and evaporation of the solvent followed by freeze drying 3 timesfrom water gave the title compound in 75% yield.

¹H-NMR (D₂O, 300 MHz): δ 0.95-1.35 (m, 5H), 1.50-2.45 (m, 15H), 3.02(bt, 1H), 3.1-3.8 (m, 7H), 4.13 (d, 1H), 4.38 (bd, 1H)

¹³C-NMR (D₂O” 75 MHz): carbonyl and guanidinecarbons: δ 154.8, 168.9,174.4

MS m/z 393 (M⁺+1)

Example 75

HOOC—CH₂—(R)Cgl-Pro-(R,S)Hig×2 HCl

(i) H—(R)Cgl-Pro-(R,S)Hig(Z)

1 g (1.6 mmole) Boc-(R)Cgl-Pro-(R,S)Hig(Z) (See Example 74 (i)) wasdissolved in 100 ml ethyl acetate saturated with HCl, and the mixturewas allowed to stand for one hour. The mixture was evaporated and theresidue was dissolved in CH₂Cl₂. The organic layer was washed twice with0.2 M NaOH-solution, dried (Na₂SO₄), filtered and evaporated to yield0.825 g (98%) of title compound.

(ii) BnOOC—CH₂—(R)Cgl-Pro-(R,S)Hig(Z)

0.442 g (0.839 mmole) H—(R)Cgl-Pro-(R,S)Hig(z), 0.256 g (1.85 mmole)K₂CO₃ and 145 μl (0.521 mmole) of bensylbromoacetate was mixed in 12 mlTHF. The mixture was stirred at 40° C. for one hour and at roomtemperature over night. After evaporation of the solvent the residue wasdissolved in CH₂Cl₂ and washed once with water and once with brine. Theorganic layer was dried (Na₂SO₄), filtered and evaporated and the crudeproduct was purified on a chromatotron (Harrison research, model 7924T)using a 2 mm silica plate with a stepwise gradient of CH₂Cl₂/MeOH (97/3,95/5, 90/10) as eluent to yield 0.165 g (29%) of the title compound.

(iii) HOOC—CH₂—(R)Cgl-Pro-(R,S)Hig×2 HCl

0.165 g (0.25 mmole) of BnOOC—CH₂—(R)Cgl-Pro-(R,S)Hig(Z) was mixed with0.050 g Pd/C (5%), 0.7 ml 1 M HCl-solution and 10 ml ethanol. Themixture was hydrogenated at atmospheric pressure for four hours.Filtration of the catalyst through cellite and evaporation of thesolvent followed by freeze drying twice from water gave 0.1 g (75%) ofthe product.

¹H-NMR (D₂O, 300 MHz): δ 1.05-1.45 (m, 5H), 1.55-2.5 (m, 15H), 3.08 (bt,1H), 3.2-4.05 (m, 9H), 4.30 (d, 1H), 4.44 (m, 1H)

¹³C-NMR (D₂O, 75 MHz): carbonyl and guanidinecarbons: δ 154.9, 167.2,169.4, 174.1

Example 76

H—(R)Cha-Pro-(R,S)Hig×2 HCl

(i) Boc-(R)Cha-Pro-(R,S)Hig(Z)

0.72 g (1.95 mmole) Boc-(R)Cha-Pro-OH (See Preparation of startingmaterials), 0.95 g (7.8 mmole) DMAP, 0.74 g (2.14 mmole) 82% pureH—(R,S)Hig(Z) (see Preparation of starting materials) in 10 ml CH₂Cl₂was added 0.486 g (2.54 mmole) of EDC and the mixture was stirred atroom temperature for 3 days. The mixture was diluted with CH₂Cl₂ andwashed with water, twice with 0.3M KHSO₄-solution and once with brineThe organic layer was dried (Na₂SO₄), filtered and evaporated and thecrude product was purified by flash chromatography using CH₂Cl₂/MeOH95/5 as eluent to yield 0.450 g (33%) of the product.

(ii) H—(R)Cha-Pro-(R,S)Hig×2 HCl

50 mg (0.078 mmole) of Boc-(R)Cha-Pro-(R,S)Hig(Z) was dissolved in 20 mlethyl acetate saturated with HCl. The mixture was allowed to stand forone hour, evaporated and the residue was dissolved in 10 ml ethanol. 20mg Pd/C (5%) and 0.3 ml 1 M HCl-solution was added and the mixture washydrogenated at atmospheric pressure for two hours. Filtration of thecatalyst through through cellite and evaporation of the solvent followedby freeze drying twice from water gave 28 mg (76%) of the titlecompound.

¹H-NMR (D₂O 300 MHz): δ 0.9-1.6 (m, 6H), 1.6-2.5 (m, 16H), 3.09 (t, 1H),3.31 (t, 1H), 3.37-3.74 (m, 4H), 3.81 (m, 1H), 4.35-4.47 (m, 2H)

¹³C-NMR (D₂O, 75 MHz): carbonyl and guanidinecarbons: δ 154.9, 169.8,174.5

Example 77

H—(R)Cgl-Aze-Rig×2 HCl

(i) Boc-(R)Cgl-Aze-Rig(Z)

To a solution of 0.50 g (1.6 mmol) of H—Rig(Z) (See Preparation ofstarting materials), 0.59 g (1.6 mmol) of Boc-(R)Cha-Aze-OH(Seepreparation of starting materials), 0.84 g (6.9 mmol) ofdimethylaminopyridine in 30 ml of acetonitrile and 5 ml ofdimethylformamide was added 0.33 g (1.7 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride. Thereaction was allowed to stir for 3 days then evaporated and partitionedbetween aqueous potassium hydrogen sulfate and methylene chloride. Themethylene chloride layer was washed with aqueous sodium bicarbonate andwater, dried (Na₂SO₄) and evaporated. The crude material was suctionfiltered through a pad of silica gel with methylene chloride/methanol9/1 to give 0.78 g (76%) of the desired compound after evaporation.

¹H NMR (300 MHz, CDCl₃): δ 0.8-1.9 (m, 27H), 2.4-2.6 (m, 2H), 2.78 (bt,2H), 3.15-3.4 (m, 2H), 3.80 (bt, 1H), 4.0-4.4 (m, 4H), 4.75 (bt, 1H),4.97 (bd, 1H), 5.08 (s, 2H), 7.1-7.4 (m, 7H), 7.74 (b, 1H).

(ii) H—(R)Cgl-Aze-Rig(Z)×2 HCl

A flask containing Boc-(R)Cgl-Aze-Rig(Z), 0.76 g (1.2 mmol), in 50 ml ofethyl acetate was cooled in an ice bath. Dry HCl was bubbled through for5 min and the solution was evaporated to give 0.74 g (100%) of thedihydrochloride as a white powder.

¹H-NMR (300 MHz, MeOD): δ 1.1-2.0 (m, 18H), 2.23 (m, 1H), 2.68 (m, 1H),3.15-3.45 (m, 4H), 3.72 (bd, 1H), 3.9-4.0 (bd, 2H), 4.27 (m, 1H), 4.39(m, 1H), 4.78 (m, 1H), 5.30 (s, 2H), 7.3-7.5 (m, 5H).

(iii) H—(R)Cgl-Aze-Rig×2 HCl

A flask containing a solution of 20 mg of H—(R)Cgl-Aze-Rig(Z) and asmall amount of 5% Pd/C was hydrogenated at atmospheric pressure for 1h. The mixture was filtered through celite and evaporated. The residuewas lyophilized with a few drops of conc. HCl added to give the product.Yield: 8 mg (52%).

¹H-NMR (300 MHz, D₂O): δ 1.1-2.0 (m, 18H), 2.37 (m, 1H), 2.75 (m, 1H),3.08 (bt, 2H), 3.39 (bt, 2H), 3.8-4.0 (m, 3H), 4.35-4.5 (m, 2H), 4.90(m, 1H).

¹³C-NMR (75.5 MHz, D₂O): guanidine and carbonyl carbons: δ 172.2, 169.4,156.4.

Example 78

HOOC—CH₂—(R)Cgl-Aze-Rig×2 HCl

(i) BnOOC—CH₂—(R)Cgl-Aze-Rig(Z)

A mixture of 0.20 g (0.33 mmol) of H—(R)Cgl-Aze-Rig(Z) (See Example 77),0.13 g of potassium carbonate, 80 mg of sodium iodide, 10 ml oftetrahydro-furane and 10 ml of acetonitrile was heated at 60° C. for 10h. The solvents were evaporated and the crude material was flashchromatographed on silica gel using methylene chloride/methanol 92/8 aseluent. Yield: 0.13 g (58%).

¹H-NMR (300 MHz, CDCl₃) δ 0.9-2.1 (m, 18H), 2.45 (m, 1H), 2.61 (m,1H),2.81 (m, 2H), 2.88 (d, 1H), 3.2-3.5 (m, 4H), 3.94 (m, 1H), 4.0-4.25 (m,3H), 4.85 (m, 1H), 5.12 (s, 2H), 5.14 (s, 2H), 6.9-7.2 (b. 2H), 7.2-7.5(m, 10H), 7.95 (m, 1H).

(ii) HOOC—CH₂—(R)Cgl-Aze-Rig×2 HCl

A mixture of 0.12 g (0.18 mmol) of BnOOC—CH₂—(R)Cgl-Aze-Rig(Z), 5 ml ofethanol, 3 drops of conc. HCl and a small amount of 5% Pd/C washydrogenated at atmospheric pressure for 1 h. The mixture was filteredthrough celite and evaporated. The residue was lyophilized in water togive 91 mg (98%) of the product.

¹H-NMR (500 MHz, D₂O): δ 1.1-1.9 (M, 17H), 2.00 (m, 1H), 2.29 (m, 1H),2.70 (m, 1H), 3.10 (m, 2H), 3.34 (t, 2H), 3.83 (bd, 2H), 3.89 (dd, 2H),4.00 (d, 1H), 4.35 (m, 2H), 4.87 (m, 1H).

¹³C NMR (125.8 MHz, D₂O): guanidine and carbonyl carbons: δ 171.8,169.6, 167.7, 156.3.

Example 79

HOOC—CH₂—CR)Cha-Pro-Rig×2 HCl

(i) Boc-(R)Cha-Pro-Rig(Z)

To a solution of 0.25 g (0.82 mmol) of4-aminoethyl-1-benzyloxycarbonylamidino piperidine(H—Rig(Z)), (Seepreparation of starting materials), 0.32 g (0.82 mmol) ofBoc-(R)Cha-Pro-OH (See Preparation of starting materials), 0.40 g (3.3mmol) of dimethylaminopyridine in 10 ml of acetonitrile and 2 ml ofdimethylformamide was added 0.165 g (0.86 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride. Thereaction was allowed to stir for 3 days then evaporated and partitionedbetween aqueous potassium hydrogen sulfate and methylene chloride. Themethylene chloride layer was washed with aqueous sodium bicarbonate andwater, dried (Na₂SO₄) and evaporated. The NMR spectrum of the crudeproduct was satisfactory and the product which contained somedimethylformamide was used in the next step without furtherpurification.

¹H-NMR (500 MHz, CDCl₃) δ 0.8-2.2 (m, 32H; thereof 1.41 (s, 9H)), 2.34(m, 1H), 2.77 (bt, 2H), 3.10 (m, 1H), 3.29 (m, 1H), 3.40 (m, 1H), 3.83(m, 1H), 4.17 (m, 2H), 4.30 (m, 1H), 4.54 (m, 1H), 5.07 (m, 1H), 5.08(s, 2H), 7.03 (m, 1H), 7.05-7.4 (m, 7H).

(ii) H—(R)Cha-Pro-Rig(Z)

A flask containing the crude product of Boc-(R)Cha-Pro-Rig(Z) in 100 mlof ethyl acetate was cooled in an ice bath. Dry HCl was bubbled throughfor 5 min and the solution was evaporated to get rid of the excess ofHCl. The product was dissolved in water and the extracted twice withethyl acetate to remove the dimethylform-amide from the previous step.The aqueous phase was made alkaline with NaHCO₃ (ag) and extracted twicewith methylene chloride. The combined organic phase was washed withwater, dried (Na₂SO₄) and evaporated. Yield: 0.37 g (81%) over twosteps.

¹H-NMR (300 MHz, CDCl₃) δ 0.8-2.4 (m, 24H), 2.82 (bt, 2H), 3.26 (m, 2H),3.42 (bq, 1H), 3.70 (m, 2H), 4.19 (m, 2H), 4.49 (bd, 1H), 5.11 (s, 2H),6.9-7.5 (m, 8H).

(iii) BnOOC—CH₂—(R)Cha-Pro-Rig(Z)

A mixture of 0.18 g (0.32 mmol) of H—(R)Cha-Pro-Rig(Z), an excess ofpotassium carbonate and 10 ml of acetonitrile was heated at 60° C. for 2h. The solvents were evaporated and the crude material was flashchromatographed on silica gel using methylene chloride/methanol 95/5 aseluent. Yield: 0.20 g (88%).

¹H-NMR (300 MHz, CDCl₃) δ 0.8-2.1 (m, 23H), 2.37 (m, 1H), 3.1-3.5 (m,7H), 4.0-4.2 (m, 2H), 4.54 (m, 1H), 5.1 (m, 4H), 6.9-7.5 (m, 13H).

(iv) HOOC—CH₂—(R)Cha-Pro-Rig×2 HCl

A mixture of 0.15 g (0.21 mmol) of BnOOC—CH₂—(R)Cha-Pro-Rig(z), 10 ml ofethanol, 4 drops of conc. HCl and a small amount of 5% Pd/C washydrogenated at atmospheric pressure for 1 h. The mixture was filteredthrough celite and evaporated. The residue was lyophilized in water togive 95 mg (64%) of the product.

¹H-NMR (500 MHz, MeOD) δ 0.85-2.1 (m, 23H), 2.30 (m, 1H), 3.10 (m, 2H),3.25 (m, 1H), 3.35 (m, 1H), 3.54 (m, 1H), 3.85-4.0 (m, 3H), 4.03 (d,1H), 4.41 (m, 1H), 4.50 (m, 1H).

¹³C-NMR (125.8 MHz, D₂O): guanidine and carbonyl carbons: δ 174.0,168.9, 168.1, 157.5.

Example 80

HOOC—CH₂—CH₂—(R)Cha-Aze-Rig×2 HCl

(i) Boc-(R)Cha-Aze-Rig(Z)

To a solution of 0.25 g (0.82 mmol) of4-aminoethyl-1-benzyloxy-cabonylamidino piperidine (H—Rig(Z)), (Seepreparation of starting materials), 0.31 g (0.86 mmol) ofBoc-(R)Cha-Aze-OH (See preparation of starting materials), 0.40 g (3.3mmol) of dimethylaminopyridine in 10 ml of acetonitrile and 2 ml ofdimethylformamide was added 0.17 g (0.86 mmol) ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride. Thereaction was allowed to stir for 3 days then evaporated and partitionedbetween aqueous potassium hydrogen sulfate and methylene chloride. Themethylene chloride layer was washed with aqueous sodium bicarbonate andwater, dried (Na₂SO₄) and evaporated. The crude product which containedsome dimethylformamide was used in the next step without furtherpurification.

¹H-NMR (500 MHz, CDCl₃) δ 0.85 (m, 1H), 0.97 (m, 1H), 1.1-1.75 (m, 26H;thereof 1.41 (s, 9H)), 1.82 (bd, 1H), 2.53 (m, 2H), 2.77 (bt, 2H), 3.25(m, 2H), 4.03 (q, 1H), 4.08 (m, 1H), 4.18 (m, 2H), 4.29 (m, 1H), 4.78(m, 1H), 4.97 (m, 1H), 5.09 (s, 2H), 7.1-7.4 (m, 7H), 7.65 (m, 1H).

(ii) H—(R)Cha-Aze-Rig(Z)

A flask containing the crude product of Boc-(R)Cha-Aze-Rig(Z) in 100 mlof ethyl acetate was cooled in an ice bath. Dry HCl was bubbled throughfor 5 min and the solution was evaporated to get rid of the excess ofHCl. The product was dissolved in water and the extracted twice withethyl acetate to remove the dimethylformamide from the previous step.The aqueous phase was made alkaline with NaHCO₃ (aq) and extracted twicewith methylene chloride. The combined organic phase was washed withwater, dried (Na₂SO₄) and evaporated. Yield: 0.31 g (70%) over twosteps.

¹H-NMR (300 MHz, CDCl₃) δ 0.8-1.9 (m, 20H), 2.48 (m, 1H), 2.73 (m, 1H),2.85 (bt, 2H), 3.25 (m, 1H), 3.35 (m, 2H), 4.05 (q, 1H), 4.1-4.25 (m,3H), 4.86 (m, 1H), 5.12 (s, 2H), 6.9-7.2 (m, 2H), 7.2-7.45 (m, 5H), 7.93(m, 1 h).

(iii) BnOOC—CH₂—CH₂—(R)Cha-Aze-Rig(Z)

A solution of 0.31 g (0.57 mmol) of H—(R)Cha-Aze-Rig(Z) and 93 mg (0.57mmol) of benzyl acrylate in 5 ml of ethanol was allowed to stand at roomtemperature for one week. It was evaporated and flash chromatographed onsilica gel using methylene chloride/methanol 94/6 as eluent. Yield: 0.20g (49%).

¹H NMR (500 MHz, CDCl₃) δ 0.8-1.0 (m, 2H), 1.1-1.8 (m, 18H), 2.48 (m,1H), 2.54 (bt, 2H), 2.68 (m, 2H), 2.81 (bt, 2H), 2.87 (m, 1H), 3.20 (m,1H), 3.25 (m, 1H), 3.31 (m, 1H), 4.04 (q, 1H), 4.1-4.2 (m, 3H), 4.84(dd, 1H), 5.05-5.15 (m, 4H), 7.0-7.5 (m, 12H), 8.03 (m, 1H).

(iv) HOOC—CH₂—CH₂—(R)Cha-Aze-Rig×2 HCl

The title compound was made and purified in the same way as described inExample 80 from 0.20 g (0.28 mmol) of BnOOC—CH₂—CH₂—(R)Cha-Aze-Rig-(Z).Yield: 30 mg (19%) of the dihydrochloride salt.

¹H-NMR (500 MHz, CDCl₃) δ 1.0-1.9 (m, 20H), 2.33 (m, 1H), 2.70 (m, 1H),2.83 (m, 2H), 3.10 (m, 2H), 3.3-3.4 (m, 4H), 3.85 (bd, 2H), 3.92 (m,rotamer), 4.14 (t, 1H), 4.17 (m, rotamer)₁ 4.31 (m, 1H), 4.46 (m, 1H),4.89 (m, 1H), 5.18 (m, rotamer).

¹³C NMR (125.8 MHz, D₂O) guanidine and carbonyl carbons: δ 175.4, 171.8,168.8, 156.3.

Example 81

HOOC—CH₂—(R)cha-Pro-(S)Itp×2 HCl

(i) Boc-(R)Cha-Pro-(S)Itp(Ts)

At roomtemperature 0.87 g (2.36 mmol) Boc-(R)Cha-Pro-OH (See preparationof startingmaterials), 0.78 g (4.72 mmol) DMAP and 0.70 g (2.36 mmol)H—(S)Itp(Ts) (See preparation of starting materials) was dissolved in 12mL acetonitrile. After stirring 20 minutes 0.59 g (3.07 mmol) EDC wasadded. After 18 hours the solvent was removed in vacuo and the residuewas dissolved in CH₂Cl₂, washed with water, citric acid (10%), KHCO₃(aq), water and dried with Na₂SO₄. Evaporation gave 1.74 g (>100% yiled(purity of about 60%)) of the desired product. Which was used in thenext step without further purification.

FAB-MS: m/z 647 (M⁺+1)

(ii) H—(R)Cha-Pro-(S)Itp(Ts)

The Boc-protecting group was removed in the same way as described forBoc-(R)Cha-Pic-(R,S)Itp(Z) (See Example 72 (ii)) to give 0.75 g (81) ofthe title compound.

FAB-MS: m/z=547 (M⁺+1)

(iii) BnOOC—CH₂—(R)Cha-Pro-(S)Itp(Ts)

0.75 g (1.37 mmol) H—(R)Cha-Pro-(S)Itp(Ts), 0.38 g (2.74 mmol) K₂CO₃ wastaken up in 15 mL acetonitrile. 0.39 g (1.65 mmol) benzylbromoacetatewas added and the mixture was stirred at 50° C. for 2 h. Evaporation ofthe solvent followed by flash chromatography using ethylacetate/methanol95/5 as eluent gave about 530 mg of the desired product.

FAB-MS: m/z=695 (M⁺+1)

(iv) HOOC—CH₂—(R)Cha-Pro-(S)Itp×2 HCl

0.53 g (0.76=mmol) BnOOC—CH₂—(R)Cha-Pro-(S)Itp(Ts) was dissolved in 15mL THF. NH₃ (g) was condensed into the flask and Na (m) was added. Thereaction was quenched after 30 min with acetic acid and the NH₃ and theTHF was evaporated. The residue was freeze dried from water and thecrude product was purified by RPLC (acetonitrile/0.1M HOAc 15/85) gave0.25 g (61%) of the desired product after freeze-drying from aqueousHCl.

¹H-NMR (500.13 MHz, D₂O); δ 0.9-2.09 (overlapping m, 20H), 2.22-2.35 (m,1H), 3.2-3.36 (m, 4H), 3.44-3.62 (overlapping m, 2H), 3.7-3.8 (m, 1H),3.87-3.99 (m, 2H), 4.33-4.48 (overlapping m, 2H).

¹³C-NMR (500.13 MHz, D₂O); carbonyl- and guanidinecarbons: δ 154.3,168.1, 169.0 and 174.2

Example 82

H—(R)Cha-Pro-(R,S)Nig×2 HCl

(i) Boc-(R)Cha-Pro-(R,S)Nig(Z)

174 mg (0.471 mmole) Boc-(R)Cha-Pro-OH (See preparation of startingmaterials), 229 mg (1.87 mmole) DMAP, 130 mg (0.471 mmole) H—(R,S)Nig(Z)(See Preparation of starting materials) was mixed in 2 ml CH₂Cl₂ and 117mg (0.61 mmole) of EDC was added and the mixture was stirred for fourdays. The mixture was diluted with CH₂Cl₂ and washed with water, twicewith 0.3 M KHSO₄-solution and once with brine The organic layer wasdried (Na₂SO₄), filtered and evaporated and the crude product waspurified twice by flash chromatography using CH₂Cl₂/MeOH 95/5 as eluentthe first time and CH₂Cl₂/MeOH 97/3 as eluent the second time to yield0.104 g (35%) of the title compound.

MS m/z 627 (M++1)

(ii) H—(R)Cha-Pro-(R,S)Nig×2 HCl

10 mg (0.016 mmole) of Boc-(R)Cha-Pro-(R,S)Nig(Z) was dissolved in 15 mlethyl acetate saturated with HCl. The mixture was allowed to stand forhalf an hour. The mixture was evaporated and the residue was dissolvedin 6 ml ethanol and 8 mg 51 Pd/C (5%) and 0.1 ml 1 M HCl-solution wasadded and the mixture was thydrogenated at atmospheric pressure for oneand a half hour. After filtration through hyflo and evaporation of thesolvent gave 4 mg of the title compound the product.

¹H-NMR (300 MHz, D₂O): δ 0.9-1.58 (m, 6H), 1.58-2.45 (m, 13H), 2.65 (m,1H), 3.19 (m, 1H), 3.34 (d, 2H), 3.4-3.73 (m, 4H), 3.82 (m, 1H),4.34-4.49 (m, 2H).

¹³C-NMR(75 MHz, D2O): carbonyl and guanidinecarbons: δ 155.1, 169.9 and174.8.

Example 83

H—(R)Pro-Phe-Pab×2 HCl

(i) Boc-(R)Pro-Phe-Pab(Z)

To a mixture of 1.2 g (3.31 mmol) Boc-(R)Pro-Phe-OH (See preparation ofstarting materials) and 1.70 g (13.91 mmol) DMAP in 40 ml CH₃CN at roomtemperature was added 0.98 g (3.35 mmol) H-Pab(Z) (See preparation ofstarting materials) dissolved in 1 ml DMF. After stirring for 2 h thereaction mixture was cooled to −18° C. and 0.66 g (3.48 mmol) EDC wasadded portion wise and the reaction was left at room temperature overnight. The solvent was evaporated and the residue was dissolved in 100ml EtOAc and washed with 1×30 ml water, 3×30 ml 0.3 M KHSO₄, 1×30 mlNa₂CO₃, 1×30 ml water and dried. Evaporation of the solvent followed byflash chromatography using CH₂Cl₂/MeOH (95/5) as eluent gave 0.691 g(38%) of the title compound.

(ii) H—(R)Pro-Phe-Pab(Z)

0.673 g Boc-(R)Pro-Phe-Pab(Z) was dissolved in 30 ml EtOAc and thesolution was saturated with HCl(g) for a few minutes (a white solidprecipitated out from the solution). The solvent and excess HCl wasevaporated and 60 ml EtOAc was added to the residue and the organicphase was washed with 2×20 ml 2 M NaOH. The washing water was extractedwith 1×25 ml EtOAc which was combined with the other EtOAc-phase and thecombined organic phase was washed with water, dried and evaporated togive 560 mg (98%) of the desired product.

¹H-NMR (500 MHz, CDCl₃): & 1.5-1.74 (m, 3H), 1.98-2.05 (m, 1H),2.78-2.85 (m, 1H), 2.90-2.96 (m, 1H), 3.0-3.2 (ABX-system centered at3.1, 2H), 3.62 (dd, 1H), 4.3-4.45 (ABX-system centered at 4.37, 2H),4.58 (q, 1H), 5.22 (s, 2H), 6.96 (bt, 1H), 7.1-7.4 (m, 10H), 7.46 (d,2H), 7.76 (d, 2H), 8.12 (d, 1H).

(iii) H—(R)Pro-Phe-Pab×2 HCl

200 mg H—(R)Pro-Phe-Pab(Z) was dissolved in 10 ml 95% EtOH and 2 ml ofwater and the mixture was hydrogenated over 5% Pd/C at atmosphericpressure for 5 h. Filtration of the catalyst and addition of 1 ml 1 MHCl followed by evaporation and freeze drying from water gave the titlecompound in 88% yield.

¹H-NMR (500 MHz, CD₃OD): δ 1.51-1.59 (m, 1H), 1.69-1.80 (m, 1H),1.87-1.97 (m, 1H), 2.19-2.29 (m, 1H), 2.90 (dd, 1H), 3.20-3.33 (m, 3H,partially hidden by the solvent peak), 4.27 (m, 1H), 4.43-4.54(AB-system centered at 4.48, 2H), 4.75-4.81 (m, 1H), 4.87 (s, 2H),7.2-7.3 (m, 5H), 7.45 (d, 2H), 7.75 (d, 2H).

¹³C-NMR (125 MHz, D₂O): amidine and carbonyl carbons: δ 166.7, 170.1 and173.4.

Example 84

HOOC—CH₂—(R)Pro-Phe-Pab×2 HCl

(i) BnOOC—CH₂—(R)Pro-Phe-Pab(Z)

To a slurry of 244 mg (0.463 mmol) H—(R)Pro-Phe-Pab(Z) (See Example 83)and 159.9 mg (1.157 mmol) K₂CO₃ in 8 ml DMF/CH₃CN (5/3) was added 127.2mg (0.555 mmol) benzylbromo acetate dissolved in 2 ml DMF and themixture was stirred at 60° C. for 1.5 h and at room temperature overnight. The solvent was evaporated and the residue was dissolved in 50 mlEtOAc, washed with 2×20 ml water and dried (Na₂SO₄). Evaporation of thesolvent followed by flash chromatography using CH₂Cl₂/MeOH (9/1) aseluent gave 176 mg (56%) of the title compound as a white solid.

¹H-NMR (300 MHz), CDCl₃): δ 1.45-1.80 (m, 3H), 2.06 (m, 1H), 2.54 (m,1H), 2.92-3.28 (m, 6H), 4.3-4.5 (ABX-system centered at δ=4.4, 2H), 4.60(dd, 1H), 5.10 (apparent s, 2H), 5.2 (apparent s, 2H), 7.1-7.4 (m, 15H),7.43 (d, 2H), 7.75 (d, 2H), 7.932 (d, 1H).

(ii) HOOC—CH₂—(R)Pro-Phe-Pab×2HCl

170 mg (0.252 mmol) of BnOOC—CH₂—(R)Pro-Phe-Pab(Z) was dissolved in 12ml EtOH/water (5/1) and hydrogenerated over 5% Pd/C at atmospheric for4.5 h. The catalyst was filtered off, the solvent evaporated and theresidue freeze dried from HCl(aq) to give the title compound.

¹H-NMR (500 MHz, CD₃OD): δ 1.62 (m, 1H), 1.82 (m, 1H), 2.08 (m, 1H),2.38 (m, 1H), 2.90 (dd, 1H), 3.25-3.35 (m, 2H), partially hidden by thesolvent peak), 3.80 (m, 1H), 4.08-4.19 (AB-system centered at 6=4.19,2H), 4.39 (m, 1H), 4.45-4.58 (AB-system centered at 6=4.50, 2H), 4.80(m, 1H), 7.20-7.35 (m, 5H), 7.45 (d, 2H), 7.75 (D, 2H).

¹³C-NMR (125 MHz, D₂O): amidine and carbonyl carbons: δ 166.8, 169.1,169.5 and 173.2.

Example 85

H—(R)Phe-Phe-Pab

(i) Boc-(R)Phe-Phe-Pab(Z)

Boc-(R)Phe-Phe-OH (16.4 mmol) (see preparation of starting materials),Pab(Z)-HCl (18.0 mmol) and 4-dimetylaminopyridine (24.6 mmol) weredissolved in 50 mL of acetonitrile. The solution was cooled to ice-watertemperature and 1-(3-dimetylaminopropyl)-3-ethylcarbodiimidehydrochloride (21.3 mmol) was added. The cooling bath was removed andthe reaction mixture was stirred over night. The solvent was thenevaporated under reduced pressure, the residue dissolved in 50 mL ofethylacetate and the resulting solution extracted with 50 mL of water.Boc-(R)Phe-Phe-Pab(Z) precipitating from the two-phase mixture wasfiltered and washed with water yielding 8.7 g (78%) after drying undervacuum at 45° C. for 24 h. ¹H NMR (200 MHz, d-CHCl₃ and d4-CH₃OH); δ8.35-7.00 (m, 19H), 4.63 (t. 1H), 4.3-4.1 (m, 1H), 3.40-2.70 (m, 6H),1.30 (s, 9H).

(ii) H—(R)Phe-Phe-Pab(Z)

Boc-(R)Phe-Phe-Pab(Z) (10.3 mmol) was slurried in 70 mL of ethylacetateand 31 mL of 3.3 M ethylacetate/HCl was added. The slurry was stirredfor 4 h after which the hydrochloride salt of H—(R)Phe-Phe-Pab(Z) wasfiltered off and washed with serveral portions of ethylacetate. The saltwas dissolved in a mixture of 50 mL of methylenechloride, 50 mL of 1 Mpotassiumcarbonate and ca 5 μL of ethanol. The organic layer wascollected and the solvent was removed under reduced pressure yielding5.0 g of H—(R)Phe-Phe-Pab(Z) (84%). ¹H NMR (200 MHz, d₆-DMSO); δ 9.1 (s,2H), 8.59 (m, 1H), 8.1 (m, 1H), 7.90 (d, 2H), 7.4-7.0 (m, 17H), 5.09 (s,2H), 4.58 (m, 1H), 4.31 (m, 2H), 3.1-2.7 (m, 4H).

(iii) H—(R)Phe-Phe-Pab(Z) (0.42 mmol) was dissolved in 10 mL oftetrahydrofuran and 1 mL of water. Palladium on charocoal (42 mg) wascharged to the solution and the mixture was hydrogenated at 45 psihydrogen pressure in a Parr shaking apparatus for 2 days. After completehydrogenolysis the mixture was diluted with methanol and the catalystwas filtered off. Evaporation of the solvents gave crudeH—(R)Phe-Phe-Pab which was purified by chromatography on neutral alumina(70-230 Mesh) eluting with methylenechloride-methanol-ammoniumhydroxide(80:20:2). Yield 76 mg of the title compound (41%). ¹H NMR (200 MHz,d₆-DMSO); δ 7.61 (d, 2H)₁ 7.4-7.0 (m, 12H), 4.64 (m, 1H), 4.44 (m, 2H),4.13 (t, 1H), 3.1-2.8 (m, 4H).

Example 86

HOOC—CO—(R)Phe-Phe-Pab

(i) MeOOC—CO—(R)Phe-Phe-Pab(Z)

H—(R)Phe-Phe-Pab(Z) (0.87 mmol) (see Example 85 (iii)) was dissolved in10 mL of tetrahydrofuran. The solution was cooled on an icewater bathand triethylamine (1.73 mmol) followed by methyloxalylchloride (0.95mmol) were added. The cooling bath was removed and the reaction mixturestirred for 18 h at ambient temperature. The reaction mixture wasdiluted with ethylacetate and extracted with water. The organic phasewas collected and the solvent was removed under reduced pressureyielding 0.45 g of MeOOC—CO—(R)Phe-Phe-Pab(Z) (78%) which was used inthe next step without further purification. TSP-MS found m/z 664(calculated for MH⁺ (C₃₇H₃₈N₅O₇) 664).

(ii) HOOC—CO—(R)Phe-Phe-Pab(Z)

MeOOC—CO—(R)Phe-Phe-Pab(Z) (0.68 mmol) was dissolved in 4 mL oftetrahydrofuran and 2 mL of water. Lithiumhydroxide (2.6 mmol) was addedand the reaction mixture was stirred at room temperature for 1.5 h.After complete hydrolysis the reaction mixture was diluted with 25 mL ofwater and acidified by addition of 0.5 mL of acetic acid. Theprecipitate was filtered and washed with several portions of wateryielding 0.40 g of crude HOOC—CO—(R)Phe-Phe-Pab(Z) after drying undervacuum at 45° C. for 24 h. The crude product was slurried in 10 mL ofethanol and 1 mL of water. The Solution was brought to reflux and theinsoluble title compound was filtered off, yielding 0.23 g ofHOOC—CO—(R)Phe-Phe-Pab(Z) (41% over two steps). ¹H NMR (200 MHz,d₆-DMSO); δ 8.62 (m, 2H), 8.41 (d, 1H), 7.89 (d, 2H), 7.4-6.9 (m, 17H),5.10 (s, 2H), 4.54 (m, 2H), 4,34 (m, 2H), 3.2-2.6 (m, 4H).

(iii) HOOC—CO—(R)Phe-Phe-Pab

HOOC—CO—(R)Phe-Phe-Pab(Z) (0.20 mmol) was slurried in 20 mL oftetrahydrofuran and 5 mL of water. Palladium on charcoal (52 mg) wascharged to the solution and the mixture was hydrogenated at 45 psihydrogen pressure in a Parr shaking apparatus for 2 days. After completehydrogenolysis the mixture was diluted with 40 mL of methanol and thecatalyst was filtered off. Evaporation of the solvents yielded 50 mg ofthe title compound (49%). ¹H NMR (200 MHz, d₆-DMSO); δ 9.2 (s), 8.78(d), 8.60 (m), 7.91 (m), 7.79 (d, 2H), 7.35-6.8 (m,12H), 4.6-4.0 (m,4H), 3.0-2.6 (m, 4H).

Example 87

HOOC—CH₂—(R)Phe-Phe-Pab

(i) BnOOC—CH₂—(R)Phe-Phe-Pab(Z)

H—(R)Phe-Phe-Pab(Z) (0.87 mmol) (see Example 85 (ii)) and potassiumcarbonate (2.6 mmol) were slurried in 10 mL of acetonitrile.Iodobenzylacetate (0.95 mmol) was added to the mixture and the solutionwas heated to 30° C. and stirred at that temperature for 2 days. Aftercomplete alkylation the solvent was removed and the residue dissolved in10 mL of ethylacetate. The solution was rapidly extracted with 10 mL ofwater and from the collected organic phase the title compoundprecipitates. BnOOC—CH₂(R)Phe-Phe-Pab(Z) was filtered off and driedunder vacuum at 45° C. for 24 h yielding 177 mgBnOOC—CH₂—(R)Phe-Phe-Pab(Z) (28%). ¹H NMR (200 MHz, CDCl₃); δ 7.79 (d,2H), 7.5-7.1 (m, 22H), 6.55 (t, 1H), 5.21 (s, 2H), 5.03 (s, 2H), 4.64(m, 1H), 4.41 (m 2H), 3.3-2.6 (m, 7H).

(ii) BnOOC—CH₂—(R)Phe-Phe-Pab(Z)

BnOOC—CH₂—(R)Phe-Phe-Pab(Z) (0.32 mmol) was slurried in 30 mL oftetrahydrofuran and 3 mL of water. Palladium on charcoal (41 mg) wascharged to the solution and the mixture was hydrogenated at 45 psihydrogen pressure in a Parr shaking apparatus for 2 days. After completehydrogenolysis the mixture was diluted with 40 mL of water and thecatalyst was filtered off. Evaporation of the solvents yielded 95 mg ofthe title compound (59%). TSP-MS found m/z 502 (calculated forMH⁺(C₂₈H₃₂N₅O₄)502).

Example 88

H—(R)Cha-Pro-Mig

(i) Boc-(R)Cha-Pro-Mig(Z)

To a stirred mixture of 0.344 g (0.93 mmol) Boc-(R)Cha-Pro-OH (seepreparation of starting materials), 0.245 g (0.93 mmol) of H-Mig(Z) (seepreparation of starting materials) and 0.227 g (1.86 mmol) of DMAP in 10mL CH₃CN was added 0.232 g (1.21 mmol) of EDC at −10° C. The reactionmixture was allowed to reach roomtemperature and left for 5 days. TheCH₃CN was evaporated and the residue was dissolved in EtOAc and washedwith H₂O, NaHCO₃ (aq) and brine. The organic layer was dried with Na₂SO₄and evaporated. The crude product was purified by flash chromatographyusing a gradient of EtOAc/MeOH, 95/5 to 90/10, as eluent to yield 0.340g (60%) of the title compund.

(ii) H—(R)Cha-Pro-Mig(Z)

0.34 g (0.55 mmol) Boc-(R)Cha-Pro-Mig(Z) was dissolved in 8 mL of EtOAcsaturated with HCl(g) and stirred for 10 min. at roomtemperature. 10 mLof a saturated solution of KOH(aq) was added dropwise. The layers wereseparated and the aqueous phase was extracted with 3×8 mL EtOAc. Theorganic layers were combined, washed with brine, dried with Na₂SO₄ andevaporated to yield 0.286 g (100%) of the title compound.

(iii) H—(R)Cha-Pro-Mig

0.050 g (0.132 mmol) of H—(R)Cha-Pro-Mig(Z) was dissolved in 3 mL MeOHand hydrogenated over 10% Pd/C at atmospheric pressure over night. Thesolution was filtered through celite and the solvent evaporated to yield0.040 g (80%) of the title compound.

¹H-NMR (500 MHz, MeOD): δ 0.92-1.02 (m, 2H), 1.18-1.47 (m, 6H),1.66-1.73 (m, 4H), 1.85-2.04 (m, 4H), 2.17-2.22 (m, 1H), 2.95-2.98 (m,1H), 3.12-3.16 (m, 1H), 3.47-3.55 (m, 2H), 3.62-3.66 (m, 1H), 3.75-3.78(m, 1H), 3.85-3.89 (m, 1H), 4.05-4.12 (m, 3H), 4.34-4.37 (m, 1H).

Signals from a minor rotamer appear at: δ 3.4, 3.7, 4.13-4.16, 4.3.

MS m/z 379 (M⁺+1)

Example 89

H—(R)Cha-Pro-Dig

(i) Boc-(R)Cha-Pro-Dig(Z)

To a stirred mixture of 0.280 g (0.76 mmol) Boc-(R)Cha-Pro-OH (seepreparation of starting materials), 0.210 g (0.76 mmol) of H-Dig(Z) (seepreparation of starting materials) and 0.186 g (1.52 mmol) of DMAP in 8mL CH₃CN was added 0.189 g (0.99 mmol) of EDC at −10° C. The reactionmixture was allowed to reach roomtemperature and left for 4 days. TheCH₃CN was evaporated and the residue was dissolved in EtOAc and washedwith H₂O, NaHCO₃ (aq) and brine. The organic layer was dried with Na₂SO₄and evaporated. The crude product was purified by flash chromatographyusing a gradient of EtOAc/MeOH, 95/5 to 90/10, as eluent to yield 0.210g (44%) of the title compund.

(ii) H—(R)Cha-Pro-Dig(Z)

0.210 g (0.33 mmol) Boc-(R)Cha-Pro-Dig(Z) was dissolved in 8=L of EtOAcsaturated with HCl(g) and stirred for 10 min. at roomtemperature. 8 mLof a saturated solution of KOH(aq) was added dropwise. The layers wereseparated and the aqueous phase was extracted with 3×8 mL EtOAc. Theorganic layers were combined, washed with brine, dried with Na₂SO₄ andevaporated to yield 0.146 g (83%) of the title compound.

(iii) H—(R)Cha-Pro-Dig

0.046 g (0.087 mmol) of H—(R)Cha-Pro-Dig(Z) was dissolved in 3 mL MeOHand hydrogenated over 10% Pd/C at atmospheric pressure over night. Thesolution was filtered through celite and the solvent evaporated to yield0.040 g (110%) of the title compound.

¹H-NMR (500 MHz, MeOD): δ 0.90-1.04 (m, 2H), 1.10-1.47 (m, 6H),1.66-1.74 (m, 4H), 1.78-2.05 (m, 4H), 2.13-2.21 (m, 1H), 2.74-2.83 (m,1H), 2.94-2.99 (m, 1H), 3.15-3.29 (m, 1H), 3.44-3.57 (m, 2H), 3.65-3.87(m, 3H), 4.07-4.25 (m, 3H), 4.35-4.39 (m, 2H).

Signals from a minor rotamer appear at: δ 4.29-4.32.

MS m/z 393 (M⁺+1)

Example 90

H—(R)Cha-Aze-Dig

(i) Boc-(R)Cha-Aze-Dig(Z)

The title compound was prepared from Boc-(R)Cha-Aze-OH and H-Dig(Z) (seepreparation of starting material) according to the procedure forBoc-(R)Cha-Pro-Dig(Z) in a yield of 0.253 g (54%).

(ii) H—(R)Cha-Aze-Dig(Z)

The title compound was prepared from Boc-(R)Cha-Aze-Dig(Z) according theprocedure for Boc-(R)Cha-Pro-Dig(Z) in a yield of 0.210 g (100%).

(iii) H—(R)Cha-Aze-Dig

0.060 g (0.117 mmol) of H—(R)Cha-Aze-Dig(Z) was dissolved in 3 mL MeOHand hydrogenated over 10% Pd/C at atmospheric pressure over night. Thesolution was filtered through celite and the solvent evaporated to yield0.042 g (95%) of the title compound.

¹H-NMR (500 MHz, MeOD): δ 0.91-1.02 (m, 2H), 1.18-1.48 (m, 6H),1.66-1.90 (m, 8H), 2.15-2.17 (m, 1H), 2.66-2.68 (m, 1H), 2.80-2.83 (m,1H), 3.14-3.29 (m, 1H), 3.39-3.44 (m, 1H), 3.72-3.80 (m, 2H), 4.01-4.04(m, 1H), 4.14-4.23 (m, 2H), 4.48-4.49 (m, 1H), 4.60-4.64 (m, 1H).

Signals from a minor rotamer appear at: δ 2.25, 2.6, 4.3, 4.67.

MS m/z 379 (M⁺+1).

EXAMPLES OF PHARMACEUTICAL PREPARATIONS

The compound according to the invention can be formulated in soliddosage forms for oral administration such as plain tablets, coatedtablets or modified release tablets. Liquid or solid-semisolid dosageforms for rectal administration. Lyophilized substance or liquids asemulsion or suspension for parenteral use. Liquid solid or semisoliddosage forms for topical administration.

In pressurized aerosols or in dry powder inhalers for oral or nasalinhalation.

Example P1

Tablets for Oral Administration

1000 tablets are prepared from the following ingredients:

Active compound 100 g Lactose 200 g Polyvinyl pyrrolidone 30 gMicrocrystalline cellulose 30 g Magnesium stearate 6 g

The active constituent and lactose are mixed with an aqueous solution ofpolyvinyl pyrrolidone. The mixture is dried and milled to form granules.The microcrystalline cellulose and then the magnesium stearate are thenadmixed. The mixture is then compressed in a tablet machine giving 1000tablets, each containing 100 mg of active constituent.

Example P2

Solution for Parenteral Administration

A solution is prepared from the following ingredients:

Active compound 5 g Sodium chloride for injection 6 g

Sodium hydroxide for pH adjustment ad pH 5-7 Water for inj. up to 1000ml

The active constituent and the sodium chloride are dissolved in thewater. The pH is adjusted with 2 M NaOH to pH 3-9 and the solution isfilled into sterile ampoules.

Example P3

Tablets for Oral Administration

1. Active compound 150 g 2. Sodium aluminium silicate 20 g 3. Paraffin120 g 4. Microcrystalline cellulose 20 g 5. Hydroxy propyl cellulose 5 g6. Sodium stearyl fumarate 3 g1-4 are mixed and an aqueous solution of 5 is added. The mixture isdried and milled and 6 is admixed. The mix is then compressed in atablet machine.

Example B6

Inhaler Powder

The active compound is micronized in a jet mill to a particle sizesuitable for inhalation (mass diameter<4 μm).

100 mg of the micronized powder is filled into a powder multidoseinhaler (Turbohaler®). The inhaler is equipped with a dosing unit whichdelivers a dose of 1 mg.

Biology

Determination of Thrombin Clotting Time (TT):

Human thrombin (T 6769, Sigma Chem Co) in buffer solution, pH 7.4, 100μl, and inhibitor solution, 100 μl, were incubated for one min. Poolednormal citrated human plasma, 100 μl, was then added and the clottingtime measured in an automatic device (KC 10, Amelung).

The clotting time in seconds was plotted against the inhibitorconcentration, and the IC₅₀TT was determined by interpolation.

IC₅₀TT is the concentration of inhibitor that doubles the thrombinclotting time for human plasma.

Determination of Activated Partial Thromboplastin Time (APTT)

APTT was determined in pooled normal human citrated plasma with thereagent PTT Automated 5 manufactured by Stago. The inhibitors were addedto the plasma (10 μl inhibitor solution to 90 μl plasma) and APTT wasdetermined in the mixture by use of the coagulation analyser KC10(Amelung) according to the instructions of the reagent producer. Theclotting time in seconds was plotted against the inhibitor concentrationin plasma and the IC₅₀APTT was determined by interpolation.

IC₅₀APTT is defined as the concentration of inhibitor in plasma thatdoubled the Activated Partial Thromboplastin Time.

Determination of Thrombin Time Ex Vivo

The inhibition of thrombin after oral administration of the compoundswere examined in conscious rats that two days prior to the experimentwere equipped with a catheter for blood sampling from the carotidartery. On the experimental day blood samples were withdrawn at fixedtimes after the administration of the compound into plastic tubescontaining 1 part sodium citrate solution (0.13 mol per L.) and 9 partsof blood. The tubes were centrifuged to obtain platelet poor plasma. Theplasma was used for determination of thrombin time as described below.

The citrated rat plasma, 100 μl, was diluted with a saline solution,0.9%, 100 μl, and plasma coagulation was started by the addition ofhuman thrombin (T 6769, Sigma Chem Co, USA) in a buffer solution, pH7.4, 100 μl. The clotting time was measured in an automatic device (KC10, Amelumg, Germany).

Determinaton of the Inhibition Constant K_(I) for Plasma Kallikrein

K_(i) determinations were made with a chromogenic substrate method, andperformed on a Cobas Bio centrifugal analyzer manufactured by Roche(Basel, Switzerland). Residual enzyme activity after incubation of humanplasma kallikrein with various concentrations of test compound wasdetermined at three different substrate concentrations, and measured aschange in optical absorbance at 405 nm and 37° C.

Human plasma kallikrein (E.C.3.4.21.34, Chromogenix AB, Mölndal,Sweden), 250 μl of 0.4 nkat/ml in buffer (0.05 mol/l Tris-HCl, pH 7.4, l0.15 adjusted with NaCl) with bovine albumin 5 g/l (cat no 810033, ICIBiochemicals Ltd, High Wycombe, Bucks, GB), was incubated for 300 s with80 μl of test compound solution in 0.15 mol/l NaCl containing albumin 10g/l. An additional 10 μl of water was supplied in this step. Then 40 μlof kallikrein substrate (S-2302, Chromogenix AB, 1.25, 2.0 or 4.0 mmol/lin water) was added together with another 20 μl of water, and theabsorbance change monitored.

K_(i) was evaluated from Dixon plots, i.e. diagrams of inhibitorconcentration versus 1/(ΔA/min), where the data for the differentsubstrate concentrations form straight lines which intercept atx=—K_(i).

Abbreviations

ABBREVIATIONS Ac = acetyl aq = aqueous Aze = Azetidine-2-carboxylic acidbetaPic = Piperidine-3-carboxylic acid Boc = tert-butyloxycarbonylBoc-Dig(Z) = 3-(N-tert-butyloxycarbonyl- aminoethyl)-1-(N-benzyloxy-carbonylamidino) azetidine Boc-Mig(Z) = 3-(N-ter-butyloxycarbonyl-aminomethyl)-1-(N-benzyloxy- carbonylamidino) azetidine Boc-Pig(Z) =4-(N-tert-butyloxycarbonyl- aminomethyl)-1-(N-benzyloxy-carbonylamidino) piperidine Boc-Pig(Z)₂ = 4-(N-tert-butyloxycarbonyl-aminomethyl)-1-(N,N′-dibenzyloxy- carbonylamidino) piperidine Brine =saturated water/NaCl solution Bn = benzyl Bu = butyl Cgl = Cyclohexylglycine Cha = β-cyclohexyl alanine CME-CDI =1-Cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonateDBU = 1,8-diazabicyclo[5.4.0]undec-7-ene DCC = dicyclohexyl carbodiimideDCU = dicyclohexyl urea DMAP = N,N-dimethyl amino pyridine DMF =dimethyl formamide DMSO = dimethyl sulphoxide EDC =1-(3-Dimetylaminopropyl)-3- ethylcarbodiimide hydrochloride Et = ethylEtOAc = ethyl acetate EtOH = ethanol Gly = glycine h = hours HCl =hydrochloric acid Hex = hexyl HOAc = acetic acid HCBt = N-hydroxybenzotriazole Hoc = Homocyclohexyl alanine Hop = Homophenyl alanine HOSu= N-hydroxysuccinimide H-Dig(Z) = 3-aminoethyl-1-(N-benzyloxycarbonyl-amidino) azetidine H-Dig = 3-aminoethyl-1-amidino azetidineH-(R,S)Hig(Z) = (3RS)-1-(N-benzyloxycarbonylamidino)- 3-aminoethylpyrrolidine H-(R,S)Hig = (3RS)-1-amidino-3-aminoethyl pyrrolidine H-Hig= 1-amidino-3-aminoethyl pyrrolidine H-(R,S)Itp(Ts) =(4RS)-1,3-diaza-2-tosylimino-4- aminoethylcyclohexane H-(R,S)Itp =(4RS)-1,3-diaza-2-imino-4- aminoethylcyclohexane H-(S)Itp(Ts) =(4S)-1,3-diaza-2-tosylimino-4- aminoethylcyclohexane H-(S)Itp =(4S)-1,3-diaza-2-imino-4-aminoethyl- cyclohexane H-Itp =1,3-diaza-2-imino-4-aminoethyl cyclohexane H-Mig(Z) =3-aminomethyl-1-(N- benzyloxycarbonylamidino) azetidine H-Mig =3-aminomethyl-1-amidino azetidine H-(R,S)Nig(Z) =(3RS)-1-(N-benzyloxycarbonylamidino)- 3-aminomethyl pyrrolidineH-(R,S)Nig = (3RS)-1-amidino-3-aminomethyl pyrrolidine H-Nig =1-amidino-3-aminomethyl pyrrolidine H-Pab = 1-amidino-4-aminomethylbenzene H-Pab(Z) = 4-aminomethyl-1-(N-benzyloxy carbonylamidino) benzeneH-Pac = 1-amidino-4-aminomethyl cyclohexane H-Pac(Z) =4-aminomethyl-1-(N-benzyloxy carbonylamidino) cyclohexane H-Pig =4-aminomethyl-1-amidino piperidine H-Pig(Z) =4-aminometyl-1-(N-benzyloxycarbonyl- amidino)-piperidine H-Pig(Z)₂ =4-aminomethyl-1-(N,N′-dibenzyloxy carbonylamidino) piperidine H-Rig(Z) =4-aminoethyl-1-(N-benzyloxy- carbonylamidino) piperidine H-Rig =4-aminoethyl-1-N-amidino piperidine Me = methyl MeOH = methanol Mpa =mega pascal Ms = mesyl NMM = N-methyl morpholine Pd/C = palladium oncharcoal Pgl = phenyl glycine Phe = phenyl alanine Pic = pipecolinicacid Pro = proline RPLC = Reverse phase high performace liquidchromathography Tf = trifluoromethylsulfonyl TFA = trifluoroacetic acidTHF = tetrahydrofuran Tic = 1-carboxy-1,2,3,4-tetrahydro- isoquinolineTs = tosyl Val = valine Z = benzyloxy carbonyl

Prefixes n, s, i and t have their usual meanings: normal, iso, sec andtertiary. The stereochemistry for the amino acids is by default (S) ifnot otherwise stated.

ABBREVIATIONS (continued, the wavy lines on the nitrogen atoms in thestructural formulas below signify the bond position of the fragment.)

1. A pharmaceutical preparation comprising an effective amount ofacetylsalicylic acid and a compound of the formula I

wherein: A¹ represents a structure of Formula IIa;

wherein: k is an integer 0, 1, 2, 3 or 4; R¹ represents H, an alkylgroup having 1 to 4 carbon atoms, or R¹¹OOC-alkyl- where the alkyl grouphas 1 to 4 carbon atoms and is optionally substituted in the positionwhich is alpha to the carbonyl group, and the alpha substituent is agroup R¹⁷—(CH₂)_(p)—, wherein p is 0, 1 or 2 and R¹⁷ is methyl, phenyl,OH, COOR¹², CONHR¹², where R¹² is H or an alkyl group having 1 to 4carbon atoms, and R¹¹ is H or an alkyl group having 1 to 4 carbon atoms,or R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or R¹represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4 carbonatoms and is optionally substituted alpha to the carbonyl with an alkylgroup having 1 to 4 carbon atoms and where R¹³ is H or an alkyl grouphaving 1 to 4 carbon atoms or —CH₂COOR¹², where R¹² is as defined above,or R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to 4carbon atoms and is optionally substituted alpha to the carbonyl with analkyl group having 1 to 4 carbon atoms and where R¹² is as definedabove, or R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,Ph(2-COOR¹²)—SO₂— where R¹² is as defined above and R¹⁴ is an alkylgroup having 1-4 carbon atoms, or R¹ represents —CO—R¹⁵, wherein R¹⁵ isan alkyl group having 1-4 carbon atoms, or R¹ represents —COOR¹⁵, whereR¹⁵ is as defined above, or R¹ represent —CO—(CH₂)_(p)—COOR², where R¹²is as defined above and p is an integer 0, 1 or 2, or R¹ represents—CH₂PO(OR¹⁶)₂, —CH₂SO₃H or —CH₂-(5-(1H)-tetrazolyl) where R¹⁸ is,individually at each occurrence, H, methyl or ethyl; R² represents H oran alkyl group having 1 to 4 carbon atoms or R²¹OOC-alkyl-, where thealkyl group has 1 to 4 carbon atoms and where R²¹ is H or an alkyl grouphaving 1 to 4 carbon atoms; R³ represents an alkyl group having 1-4carbon atoms, and the alkyl group may be optionally substituted by oneor more fluorine atoms, or R³ represents a cyclopentyl, cyclohexyl- or aphenyl group which may be optionally substituted with an alkyl grouphaving 1 to 4 carbon atoms, or R³ represents a phenyl group substitutedwith a OR³¹ group, where R³¹ is H or an alkyl group having 1 to 4 carbonatoms and k is 0, 1, or R³ represents a 1-naphthyl or 2-naphthyl groupand k is 0, 1, or R³ represent a cis- or trans-decalin group and k is 0,1, or R³ represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl which may beoptionally substituted with a OR³¹ group, where R³¹ is as defined aboveand k is 0, 1, or R³ represents Si(Me)₃ or CH(R³²)₂ wherein R³² is acyclohexyl- or a phenyl group; A² represents a structure of formulaIIIa,

wherein: Y represents a methylene group, or Y represents an ethylenegroup and the resulting 5-membered ring may be optionally substituted byone or two fluorine atoms, a hydroxy group or an oxo group in position4, or may be optionally unsaturated, or Y represents —CH₂—O—, —CH₂—S—,—CH₂—SO—, with the heteroatom functionality in position 4, or Yrepresents a n-propylene group and the resulting 6-membered ring may beoptionally substituted in position 5 by one fluorine atom, a hydroxygroup or an oxo group, may be optionally substituted by two fluorineatoms in one of positions 4 or 5 or may be optionally unsaturated inposition 4 and 5, or may be optionally substituted in position 4 by analkyl group with 1 to 4 carbon atoms, or Y represents —CH₂—O—CH₂—,—CH₂—S—CH₂—, —CH₂—SO—CH₂—, or Y represent —CH₂—CH₂—CH₂—CH₂—; R⁵represents H or an alkyl group having 1 to 4 carbon atoms, or R⁵represents —(CH₂)P—COOR⁵¹, where p is 0, 1 or 2 and R⁵¹ is H or an alkylgroup having 1 to 4 carbon atoms; n is an integer 0, 1, 2, 3 or 4; Brepresents a group of Formula IVb,

wherein: X⁵ represents C(NH)—NH₂; R⁶ is H or an alkyl group having 1-4carbon atoms; either the compound as such or stereoisomers thereof or inthe form of a physiologically acceptable salt, in association with apharmaceutical carrier.
 2. A pharmaceutical preparation comprising aneffective amount of acetylsalicylic acid and a compound of the formula

wherein: A¹ represents a structure of Formula IIa;

wherein: k is an integer 0, 1, 2, 3 or 4; R¹ represents R¹¹OOC-alkyl-,where the alkyl group has 1 to 4 carbon atoms and is optionallysubstituted in the position which is alpha to the carbonyl group, andthe alpha substituent is a group R¹⁷—(CH₂)_(p)—, wherein p is 0, 1 or 2and R¹⁷ is COOR¹², CONHR¹², where R¹² is H, an alkyl group having 1 to 4carbon atoms or a benzyl group, and R¹¹ is H or an alkyl group having 1to 4 carbon atoms, or a benzyl group, or R¹ representsPh(4-COOR¹²)—CH₂—, where R¹² is as defined above, or R¹ representsR¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4 carbon atoms and isoptionally substituted alpha to the carbonyl with an alkyl group having1 to 4 carbon atoms and where R¹³ is H or an alkyl group having 1 to 4carbon atoms or —CH₂COOR¹², where R¹² is as defined above, or R¹represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to 4carbon atoms and is optionally substituted alpha to the carbonyl with analkyl group having 1 to 4 carbon atoms and where R¹² is as definedabove, or R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,Ph(2-COOR¹²)—SO₂—where R¹² is as defined above and R¹⁴ is an alkyl grouphaving 1-4 carbon atoms, or R¹ represents —CO—R⁵, wherein R¹⁵ is analkyl group having 1-4 carbon atoms, or R¹ represents —CO—OR¹⁵, whereR¹⁵ is as defined above, or R¹ represent —CO—(CH₂)_(p)—COOR², where R¹²is as defined above and p is an integer 0, 1 or 2, or R² represents H oran alkyl group having 1 to 4 carbon atoms or R²¹OOC-alkyl-, where thealkyl group has 1 to 4 carbon atoms and where R²¹ is H or an alkyl grouphaving 1 to 4 carbon atoms or a benzyl group; R³ represents an alkylgroup having 1-4 carbon atoms, and the alkyl group may be optionallysubstituted by one or more fluorine atoms, or R³ represents acyclopentyl, cyclohexyl- or a phenyl group which may be optionallysubstituted with an alkyl group having 1 to 4 carbon atoms, or R³represents a phenyl group substituted with a OR³¹ group, where R³¹ is Hor an alkyl group having 1 to 4 carbon atoms and k is 0, 1, or R³represents a 1-naphthyl or 2-naphthyl group and k is 0, 1, or R³represent a cis- or trans-decalin group and k is 0, 1, or R³ represents4-pyridyl, 3-pyrrolidyl or 3-indolyl which be optionally substitutedwith a OR³¹ group, where R³¹ is as defined above and k is 0, 1, or R³represents Si(Me)₃ or CH(R³²)₂, wherein R³² is a cyclohexyl- or a phenylgroup; A² represents a structure of Formula IIIa

wherein: Y represents a methylene group, or Y represents an ethylenegroup and the resulting 5-membered ring may be optionally substituted byone or two fluorine atoms, a hydroxy group or an oxo group in position4, or may be optionally unsaturated, or Y represents —CH₂—O—, —CH₂—S—,—CH₂—SO—, with the heteroatom functionality in position 4, or Yrepresents a n-propylene group and the resulting 6-membered ring may beoptionally substituted in position 5 by one fluorine atom, a hydroxygroup or an oxo group, may be optionally substituted by two fluorineatoms in one of positions 4 or 5 or may be optionally unsaturated inposition 4 and 5, or may be optionally substituted in position 4 by analkyl group with 1 to 4 carbon atoms, or Y represents —CH₂—O—CH₂—,—CH₂—S—CH₂—, —CH₂—SO—CH₂—, or Y represent —CH₂—CH₂—CH₂—CH₂—; R⁵represents H or an alkyl group having 1 to 4 carbon atoms, or R⁵represents —(CH₂)_(p)—COOR⁵¹, where p is 0, 1 or 2 and R⁵¹ is H or analkyl group having 1 to 4 carbon atoms; n is an integer 0, 1, 2, 3 or 4;B represents a group of Formula IVb

wherein: X⁵ represents C(NH)—NH₂; R⁶ is H or an alkyl group having 1-4carbon atoms; D is Z or (Z)₂; Z is a benzyloxy carbonyl group; eitherthe compound as such or stereoisomers thereof or in the form of aphysiologically acceptable salt, in association with a pharmaceuticalcarrier.
 3. A pharmaceutical preparation according to claim 1 or 1wherein R¹ represents R¹¹OOC-alkyl-, where the alkyl group has 1 to 4carbon atoms and R¹¹ is H.
 4. A pharmaceutical preparation according toclaim 1 or 2 wherein B is a structure of formula IVb, wherein X⁵ isC(NH)—NH₂ and R⁶ is H and n is
 1. 5. A pharmaceutical preparationaccording to claim 1 or 2 in which n is 1, A¹ is a structure of formulaIIa wherein k is 0 or 1, R¹ represents R¹¹OOC-alkyl-, where the alkylgroup has 1 to 4 carbon atoms, R² represents H, R³ represents acyclohexyl group, A² represents a structure of formula lla wherein Yrepresents a methylene group, an ethylene group, or a n-propylene groupand the resulting 6-membered ring may be optionally substituted inposition 4 by an alkyl group with 1 to 4 carbon atoms, R⁵ represents H,B represents a structure of formula IVb wherein X⁵ represents C(NH)—NH₂and R⁶ is H.
 6. A pharmaceutical preparation comprising an effectiveamount of acetylsalicylic acid and a compound selected fromHOOC—CH₂—(R)Cgl-Aze-Pab HOOC—CH₂—CH₂—(R)Cgl-Aze-PabHOOC—CH₂—(R)Cgl-Pro-Pab HOOC—CH₂—CH₂—(R)Cgl-Pro-Pab(HOOC—CH₂)₂—(R)Cgl-Pro-Pab H—(R)Cgl-Pic-PabHOOC—CH₂—(R,S)CH(COOH)—(R)Cgl-Pic-Pab H—(R)Cha-Aze-PabHOOC—CH₂—(R)Cha-Aze-Pab HOOC—CH₂—(R,S)CH(COOH)—(R)Cha-Aze-PabHOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Aze-Pab/a HOOC—CH₂—(R orS)CH(COOH)—(R)Cha-Aze-Pab/b HOOC—CH₂—CH₂—(R)Cha-Aze-PabHOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab H—(R)Cha-Pro-PabHOOC—CH₂—(R)Cha-Pro-Pab HOOC—CH₂-(Me)(R)Cha-Pro-PabHOOC—CH₂—CH₂—(R)Cha-Pro-Pab HOOC—CH₂—CH₂—(Me)(R)Cha-Pro-Pab HOOC—CH₂—(Ror S)CH(COOH)—(R)Cha-Pro-Pab/a HOOC—CH₂—(R orS)CH(COOH)—(R)Cha-Pro-Pab/b HOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-PabEtOOC—CH₂—CH₂—CH₂—(R)Cha-Pro-Pab Ph(4-COOH)—SO₂—(R)Cha-Pro-PabH—(R)Cha-Pic-Pab HOOC—CH₂—(R)Cha-Pic-Pab HOOC—CH₂—(R orS)CH(COOH)—(R)Cha-Pic-Pab/a HOOC—CH₂—(R or S)CH(COOH)—(R)Cha-Pic-Pab/bHOOC—CH₂—CH₂—(R)Cha-Pic-Pab HOOC—CO—(R)Cha-Pic-PabHOOC—CH₂—CO—(R)Cha-Pic-Pab Me—OOC—CH₂—CO—(R)Cha-Pic-PabH₂N—CO—CH₂—(R)Cha-Pic-Pab Boc-(R)Cha-Pic-Pab Ac—(R)Cha-Pic-PabMe—SO₂—(R)Cha-Pic-Pab H—(R)Hoc-Aze-Pab HOOC—CH₂—CH₂—(R)Hoc-Aze-PabHOOC—CH₂—(R,S)CH(COOH)—(R)Hoc-Pro-Pab HOOC—CH₂—(R)Hoc-Pic-Pab(HOOC—CH₂)₂—(R)Hoc-Pic-Pab H—(R)Cgl-Aze-PabHOOC—(R,S)CH(Me)—(R)Cha-Pro-Pab MeOOC—CH₂—(R)Cgl-Aze-PabEtOOC—CH₂—(R)Cgl-Aze-Pab ^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab either as such or a stereoisomer thereofor in the form of a physiologically acceptable salt, in association witha pharmaceutical carrier.
 7. A pharmaceutical preparation comprising aneffective amount of acetylsalicylic acid and a compound selected fromHOOC—CH₂—(R)Cgl-Aze-Pab HOOC—CH₂—CH₂—(R)Cha-Aze-PabHOOC—CH₂—(R)Cha-Pro-Pab HOOC—CH₂—CH₂—(R)Cha-Pro-PabHOOC—CH₂—(R)Cha-Pic-Pab EtOOC—CH₂—(R)Cgl-Aze-Pab either as such or astereoisomer thereof or in the form of a physiologically acceptablesalt, in association with a pharmaceutical carrier.
 8. A pharmaceuticalpreparation comprising an effective amount of acetylsalicylic acid and acompound selected from BnOOC—CH₂—(R)Cgl-Aze-Pab(Z)BnOOC—CH₂—CH₂—(R)Cgl-Aze-Pab (Z) BnOOC—CH₂—(R)Cgl-Pro-Pab(Z)BnOOC—CH₂—CH₂—(R)Cgl-Pro-Pab(Z) (BnOOC—CH₂)₂—(R)Cgl-Pro-Pab(Z)BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cgl-Pic-Pab(Z) BnOOC—CH₂—(R)Cha-Aze-Pab (Z)BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Aze-Pab(Z) BnOOC—CH₂—(R orS)CH(COOBn)—(R)Cha-Aze-Pab(Z)/a BnOOC—CH₂—(R orS)CH(COOBn)—(R)Cha-Aze-Pab(Z)/b BnOOC—CH₂—CH₂—(R)Cha-Aze-Pab(Z)BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Aze-Pab(Z) BnOOC—CH₂—(R)Cha-Pro-Pab(Z)BnOOC—CH₂-(Me)(R)Cha-Pro-Pab(Z) BnOOC—CH₂—CH₂—(R)Cha-Pro-Pab(Z)BnOOC—CH₂—CH₂-(Me)(R)Cha-Pro-Pab(Z)BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pro-Pab(Z)BnOOC—CH₂—NH—CO—CH₂—(R)Cha-Pro-Pab (Z) Ph(4-COOH)—SO₂—(R)Cha-Pro-Pab(Z)Boc-(R)Cha-Pic-Pab(Z) BnOOC—CH₂—(R)Cha-Pic-Pab(Z)BnOOC—CH₂—(R,S)CH(COOBn)—(R)Cha-Pic-Pab(Z)BnOOC—CH₂—CH₂—(R)Cha-Pic-Pab(Z) EtOOC—CO—(R)Cha-Pic-Pab(Z)MeOOC—CH₂—CO—(R)Cha-Pic-Pab(Z) H₂N—CO—CH₂—(R)Cha-Pic-Pab (Z)Ac—(R)Cha-Pic-Pab (Z) Me—SO₂—(R)Cha-Pic-Pab(Z)BnOOC—CH₂—CH₂—(R)Hoc-Aze-Pab(Z)BnOOC—CH₂—(R,S)CH(COOBn)—(R)Hoc-Pro-Pab(Z) BnOOC—CH₂—(R)Hoc-Pic-Pab(Z)(BnOOC—CH₂)₂—(R)Hoc-Pic-Pab(Z) BnOOC—(R,S)CH (Me)—(R)Cha-Pro-Pab(Z)MeOOC—CH₂—(R)Cgl-Aze-Pab(Z) EtOOC—CH₂—(R)Cgl-Aze-Pab(Z)^(n)BuOOC—CH₂—(R)Cgl-Aze-Pab(Z) ^(n)HexOOC—CH₂—(R)Cgl-Aze-Pab(Z) eitheras such or a stereoisomer thereof or in the form of a physiologicallyacceptable salt, in association with a pharmaceutical carrier.
 9. Apharmaceutical preparation comprising an effective amount ofacetylsalicylic acid and a compound selected fromBnOOC—CH₂—(R)Cgl-Aze-Pab(Z) BnOOC—CH₂—(R)Cha-Pro-Pab(Z)BnOOC—CH₂—(R)Cha-Pic-Pab(Z) EtOOC—CH₂—(R)Cgl-Aze-Pab(Z) either as suchor a stereoisomer thereof or in the form of a physiologically acceptablesalt, in association with a pharmaceutical carrier.
 10. A pharmaceuticalpreparation comprising an effective amount of acetylsalicylic acid andthe compound HOOC—CH₂—(R)Cgl-Aze-Pab or a stereoisomer thereof or aphysiologically acceptable salt thereof, in association with apharmaceutical carrier.
 11. A pharmaceutical preparation comprising aneffective amount of acetylsalicylic acid and the compoundEtOOC—CH₂—(R)Cgl-Aze-Pab or a stereoisomer thereof or a physiologicallyacceptable salt thereof, in association with a pharmaceutical carrier.12. A pharmaceutical preparation comprising an effective amount ofacetylsalicylic acid and the compound HOOC—CH₂—(R)Cgl-Aze-Pab, inassociation with a pharmaceutical carrier.
 13. A pharmaceuticalpreparation comprising an effective amount of acetylsalicylic acid andthe compound EtOOC—CH₂—(R)Cgl-Aze-Pab, in association with apharmaceutical carrier.
 14. A method for inhibiting thrombin in a mammalin need of such inhibition, which comprises administering to the mammalan effective amount of a pharmaceutical preparation as claimed inclaim
 1. 15. A method for inhibiting thrombin in a mammal in need ofsuch inhibition, which comprises administering to the mammal aneffective amount of a pharmaceutical preparation as claimed in claim 2.16. A method for inhibiting thrombin in a mammal in need of suchinhibition, which comprises administering to the mammal an effectiveamount of a pharmaceutical preparation as claimed in claim
 10. 17. Amethod for inhibiting thrombin in a mammal in need of such inhibition,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 11. 18. A method forinhibiting thrombin in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 12. 19. A method forinhibiting thrombin in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 13. 20. A method oftreatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 1. 21. A method oftreatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 2. 22. A method oftreatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 10. 23. A method oftreatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 11. 24. A method oftreatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 12. 25. A method oftreatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 13. 26. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 1. 27. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 2. 28. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 10. 29. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 11. 30. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 12. 31. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of apharmaceutical preparation as claimed in claim
 13. 32. A combinationcomprising (a) acetylsalicylic acid and (b) a compound of the formula I

wherein: A¹ represents a group of Formula IIa;

wherein: k is an integer 0, 1, 2, 3 or 4; R¹ represents H, an alkylgroup having 1 to 4 carbon atoms, or R¹¹OOC-alkyl- where the alkyl grouphas 1 to 4 carbon atoms and is possibly substituted in the positionwhich is alpha to the carbonyl group, and the alpha substituent is agroup R¹⁷—(CH₂)_(p)—, wherein p is 0, 1 or 2 and R¹⁷ is methyl, phenyl,OH, COOR¹², CONHR¹², where R¹² is H or an alkyl group having 1 to 4carbon atoms, and R¹¹ is H or an alkyl group having 1 to 4 carbon atoms,or R¹ represents Ph(4-COOR¹²)—CH₂—, where R¹² is as defined above, or R¹represents R¹³—NH—CO-alkyl-, where the alkyl group has 1 to 4 carbonatoms and is possibly substituted alpha to the carbonyl with an alkylgroup having 1 to 4 carbon atoms and where R¹³ is H or an alkyl grouphaving 1 to 4 carbon atoms or —CH₂COOR¹², where R¹² is as defined above,or R¹ represents R¹²OOC—CH₂—OOC-alkyl-, where the alkyl group has 1 to 4carbon atoms and is possibly substituted alpha to the carbonyl with analkyl group having 1 to 4 carbon atoms and where R¹² is as definedabove, or R¹ represents R¹⁴SO₂—, Ph(4-COOR¹²)—SO₂—, Ph(3-COOR¹²)—SO₂—,Ph(2-COOR¹²)—SO₂— where R¹² is as defined above and R¹⁴ is an alkylgroup having 1-4 carbon atoms, or R¹ represents —CO—R¹⁵, wherein R¹⁵ isan alkyl group having 1-4 carbon atoms, or R¹ represents —COOR¹⁵, whereR¹⁵ is as defined above, or R¹ represent —CO—(CH₂)_(p)—COOR², where R¹²is as defined above and p is an integer 0, 1 or 2, or R¹ represents—CH₂PO(OR¹⁶)₂, —CH₂SO₃H or —CH₂-(5-(1H)-tetrazolyl) where R¹⁶ is,individually at each occurrence, H, methyl or ethyl; R² represents H oran alkyl group having 1 to 4 carbon atoms or R²¹OOC-alkyl-, where thealkyl group has 1 to 4 carbon atoms and where R²¹ is H or an alkyl grouphaving 1 to 4 carbon atoms; R³ represents an alkyl group having 1-4carbon atoms, and the alkyl group may or may not carry one or morefluorine atoms, or R³ represents a cyclopentyl, cyclohexyl- or a phenylgroup which may or may not be substituted with an alkyl group having 1to 4 carbon atoms, or R³ represents a phenyl group substituted with aOR³¹ group, where R³′ is H or an alkyl group having 1 to 4 carbon atomsand k is 0, 1, or R³ represents a 1-naphthyl or 2-naphthyl group and kis 0, 1, or R³ represent a cis- or trans-decalin group and k is 0, 1, orR³ represents 4-pyridyl, 3-pyrrolidyl or 3-indolyl which may or may notbe substituted with a OR³¹ group, where R³¹ is as defined above and k is0, 1, or R³ represents Si(Me)₃ or CH(R³²)₂ wherein R³² is a cyclohexyl-or a phenyl group; A² represents a group of formula IIIa

wherein: Y represents a methylene group, or Y represents an ethylenegroup and the resulting 5-membered ring may or may not carry one or twofluorine atoms, a hydroxy group or an oxo group in position 4, or may ormay not be unsaturated, or Y represents —CH₂—O—, —CH₂—S—, —CH₂—SO—, withthe heteroatom functionality in position 4, or Y represents an-propylene group and the resulting 6-membered ring may or may not carryin position 5 one fluorine atom, a hydroxy group or an oxo group, carrytwo fluorine atoms in one of positions 4 or 5 or be unsaturated inposition 4 and 5, or carry in position 4 an alkyl group with 1 to 4carbon atoms, or Y represents —CH₂—O—CH₂—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—, orY represent —CH₂—CH₂—CH₂—CH₂—; R⁵ represents H or an alkyl group having1 to 4 carbon atoms, or R⁵ represents —(CH₂)_(p)—COOR⁵, where p is 0, 1or 2 and R⁵¹ is H or an alkyl group having 1 to 4 carbon atoms; n is aninteger 0, 1, 2, 3 or 4; B represents a group of Formula IVb

wherein: X⁵ represents C(NH)—NH₂; R⁶ is H or an alkyl group having 1-4carbon atoms; either the compound as such or stereoisomers thereof or inthe form of a physiologically acceptable salt.
 33. A combinationcomprising (a) acetylsalicylic acid and (b) the compoundHOOC—CH₂—(R)Cgl-Aze-Pab or a stereoisomer thereof or a physiologicallyacceptable salt thereof.
 34. A combination comprising (a)acetylsalicylic acid and (b) the compound EtOOC—CH₂—(R)Cgl-Aze-Pab or astereoisomer thereof or a physiologically acceptable salt thereof.
 35. Acombination comprising (a) acetylsalicylic acid and (b) the compoundHOOC—CH₂—(R)Cgl-Aze-Pab.
 36. A combination comprising (a)acetylsalicylic acid and (b) the compound EtOOC—CH₂—(R)Cgl-Aze-Pab. 37.A combination as claimed in claim 32 which comprises a kit of partscomprising components (a) and (b).
 38. A combination product as claimedin claim 32, in which components (a) and (b) are suitable forsequential, separate and/or simultaneous use in the treatment of acondition where inhibition of thrombin is required.
 39. A combinationproduct as claimed in claim 38 wherein component (a) is combined withcomponent (b).
 40. A method for inhibiting thrombin in a mammal in needof such inhibition, which comprises administering to the mammal aneffective amount of a combination as claimed in claim
 32. 41. A methodfor inhibiting thrombin in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of acombination as claimed in claim
 33. 42. A method for inhibiting thrombinin a mammal in need of such inhibition, which comprises administering tothe mammal an effective amount of a combination preparation as claimedin claim
 34. 43. A method for inhibiting thrombin in a mammal in need ofsuch inhibition, which comprises administering to the mammal aneffective amount of a combination as claimed in claim
 35. 44. A methodfor inhibiting thrombin in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of acombination as claimed in claim
 36. 45. A method for inhibiting thrombinin a mammal in need of such inhibition, which comprises administering aneffective amount of components (a) and (b) of a combination as claimedin claim 32 separately, sequentially or simultaneously to the mammal.46. A method of treatment or prophylaxis of thrombosis and ofhypercoagulability in blood and tissues in a mammal in need of suchtreatment or prophylaxis, which comprises administering to the mammal aneffective amount of a combination as claimed in claim
 32. 47. A methodof treatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering to the mammal an effective amount of acombination as claimed in claim
 33. 48. A method of treatment orprophylaxis of thrombosis and of hypercoagulability in blood and tissuesin a mammal in need of such treatment or prophylaxis, which comprisesadministering to the mammal an effective amount of a combination asclaimed in claim
 34. 49. A method of treatment or prophylaxis ofthrombosis and of hypercoagulability in blood and tissues in a mammal inneed of such treatment or prophylaxis, which comprises administering tothe mammal an effective amount of a combination as claimed in claim 35.50. A method of treatment or prophylaxis of thrombosis and ofhypercoagulability in blood and tissues in a mammal in need of suchtreatment or prophylaxis, which comprises administering to the mammal aneffective amount of a combination as claimed in claim
 36. 51. A methodof treatment or prophylaxis of thrombosis and of hypercoagulability inblood and tissues in a mammal in need of such treatment or prophylaxis,which comprises administering an effective amount of components (a) and(b) of a combination as claimed in claim 22 separately, sequentially orsimultaneously to the mammal.
 52. A method for inhibiting kininogenasesin a mammal in need of such inhibition, which comprises administering tothe mammal an effective amount of a combination as claimed in claim 32.53. A method for inhibiting kininogenases in a mammal in need of suchinhibition, which comprises administering to the mammal an effectiveamount of a combination as claimed in claim
 33. 54. A method forinhibiting kininogenases in a mammal in need of such inhibition, whichcomprises administering to the mammal an effective amount of acombination as claimed in claim
 34. 55. A method for inhibitingkininogenases in a mammal in need of such inhibition, which comprisesadministering to the mammal an effective amount of a combination asclaimed in claim
 35. 56. A method for inhibiting kininogenases in amammal in need of such inhibition, which comprises administering to themammal an effective amount of a combination as claimed in claim
 36. 57.A method for inhibiting kininogenases in a mammal in need of suchinhibition, which comprises administering an effective amount ofcomponents (a) and (b) of a combination as claimed in claim 32separately, sequentially or simultaneously to the mammal.